Introduction to Light Microscopy: Tips and Tricks for Beginners [1st ed.] 978-3-030-05392-5;978-3-030-05393-2

Table of contents :
Front Matter ....Pages i-xxi
Introduction (Dee Lawlor)....Pages 1-9
The Advantages of Learning Traditional Microscopy (Dee Lawlor)....Pages 11-16
Buying a Microscope (Dee Lawlor)....Pages 17-23
The Science of Light (Dee Lawlor)....Pages 25-37
Introduction to Lenses (Dee Lawlor)....Pages 39-47
The Anatomy of the Microscope (Dee Lawlor)....Pages 49-55
The Eyepiece (Dee Lawlor)....Pages 57-63
The Objective (Dee Lawlor)....Pages 65-79
The Stage (Dee Lawlor)....Pages 81-86
The Condenser and Diaphragm (Dee Lawlor)....Pages 87-94
Light Sources (Dee Lawlor)....Pages 95-103
Choosing the Right Technique (Dee Lawlor)....Pages 105-114
Sample Prep (Dee Lawlor)....Pages 115-126
Image Capture (Dee Lawlor)....Pages 127-137
Troubleshooting (Dee Lawlor)....Pages 139-154
Back Matter ....Pages 155-164

Citation preview

Dee Lawlor

Introduction to Light Microscopy Tips and Tricks for Beginners

Introduction to Light Microscopy

Dee Lawlor

Introduction to Light Microscopy Tips and Tricks for Beginners

Dee Lawlor Aberdeen, Aberdeenshire, UK

ISBN 978-3-030-05392-5    ISBN 978-3-030-05393-2 (eBook) https://doi.org/10.1007/978-3-030-05393-2 © Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover illustration: Kateryna Kon/shutterstock This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

For my mum, who is the reason I became a scientist. Sorry for all the weird stuff you used to find in the fridge.

Preface

I remember my first microscope—I still have it, in fact. It was a toy microscope that I got for Christmas when I was in primary school. This, of course, was back in the day when children’s toys still came with scalpels and chemicals. I had hacked up an onion and lashed on some methylene blue. I squished down the coverslip and spilled the stain all over my mother’s chopping board (note: methylene blue does not come out of wooden chopping boards). Under the battery light of my little microscope, there they were—the first cells I had ever seen. Throughout my education, I think I had only had two or three classes on microscopy. My knowledge of sample prep was pretty dire too. Not through any fault of my educators, but purely because—as a student—the goal was to get to the final image. The journey of how to get there didn’t seem that important. My journey into professional microscopy was born from panic. I had graduated my degree in zoology from the University of Aberdeen in 2010, after which I moved home to Dublin and was accepted into UCD to study my great love, evolutionary biology. Two weeks into my course, panic struck! At that time, Ireland was deep in recession and all thoughts were on what job you could get after graduation. Unfortunately, there were not many wanted ads looking for an evolutionary biologist. I hastily tore through the universities prospectus and found an MSc course in microscopy and image analysis. This was an entirely new subject to me. Like all biologists I had used microscopes, but like many people, I had never given them much thought. I realised that microscopy was a skill, and it is a skill that is used in all areas of science. In the world of research, having good lab skills is invaluable. I figured I couldn’t lose, and in fact, I gained so much more than I ever expected. Aberdeen, UK

Dee Lawlor vii

Acknowledgements

A huge thank you to my partner in life, crime and adventure, Blair, who had a never-ending supply of pep talks and for the many hours he spent on CAD drawings for me.  A special thank you to Mr. Kevin MacKenzie, Dr. Tyler Stevenson, and Mr. Karl Gaff for contributing images.  Thank you to my wonderful family and friends for their help and support: Jenny, Charlie, Eile, Adrienne, Brian, Dara, Robyn, Marie, Linda D. John, Sinead, Caitriona Nigel, Claire, Jamie, Les, TC, Linda N. and Lily. Thank you to my editors Markus Spaeth and Srinivasan Manavalan for their guidance through my first publishing experience. Finally, thank you to Prof. Jeremy Simpson, who showed me the light.

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About the Book

Microscopes are one of the most utilised and most ignored machines in the lab. Being easy and reliable to use, many researchers and scientists don’t need to put too much thought into what they are doing or the machine they are using. That is, of course, until something goes wrong. The goal of this book is to give microscope users a solid grounding in the technology they are using. With a strong knowledge of the basics, the user will be confident in approaching higher end systems and will be an invaluable resource in any lab. Over the next few chapters we shall break the microscope down into its constituent parts, discuss how the part is assembled, how it works, and how to use it best. To cover as many bases as possible I have included traditional upright microscopes, geology microscopes, and inverted microscopes. I have included guides on sample preparation for brightfield microscopy and more general chapters such as consumables and what to consider when buying a microscope. As much as possible I have used clear and simple language. Where technical terms are required I have provided straightforward explanations. The troubleshooting chapter will cover a few extra notes and methods to assist in smooth imaging, but with a good introductory knowledge to microscopes, the user will be capable of troubleshooting many issues themselves. One of the best things about microscopes is that they are accessible to everyone. No matter what the budget or requirement, there is a microscope that fits. Everyone has equal access to this microworld, and my goal for this book is to give every microscope user a clear field of view. Whether you are an undergrad in university, a teacher trying to introduce young students to science, or an armchair adventurer, I promise you this—once you take your first look down the microscope, there will be no looking back. xi

Contents

1 Introduction  1 1.1 History   2 1.2 The Importance of Glass in Microscopy   6 1.3 What Classifies as a Microscope?   7 1.4 Why We Find Microscopes Appealing   8 References  9 2 The Advantages of Learning Traditional Microscopy 11 2.1 Skill Acquisition  12 2.2 Enjoying Knowledge   12 2.3 Budget-Friendly  13 2.4 Low Training Requirement  13 2.5 Robust  14 2.6 Reliable  14 2.7 Easy Troubleshooting  15 2.8 The Technology Doesn’t Age  15 2.9 Easy to Move  15 References 16 3 Buying a Microscope 17 3.1 Decide What Is Needed  18 3.1.1 Who Is Going to Be Using the Microscope?  18 3.1.2 What Type of Samples Are the Users Working with? 19

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3.1.3 How Often Is the Microscope Going to Be Used?  19 3.1.4 Where Is the Microscope Going to Be Used?  19 3.1.5 Will It Stay Set Up or Will It Need to Be Packed Up After Each Use?  20 3.1.6 Does the User Require Image Capture?  20 3.2 Budget and Where to Invest  20 3.3 Microscopes for Children  21 3.4 Buying a Second-Hand Microscope  21 4 The Science of Light 25 4.1 What Is Light?  26 4.2 The Basics of Light  26 4.3 Colours of Light  28 4.4 Movement of Light  29 4.4.1 Refraction  29 4.4.2 Reflection  30 4.4.3 Absorption and Emission  32 4.4.4 Scatter  32 4.5 Photosensitivity: How We Perceive Light  33 4.5.1 The Evolution of Vision  33 4.5.2 Photosensitive Cells  34 4.5.3 Human Vision  34 4.5.4 How We See  35 4.5.5 Colour Vision  35 4.5.6 Variation of Vision Between Individuals  36 4.5.7 Colour-Blindness  36 4.5.8 Variation in Physiology  37 References 37 5 Introduction to Lenses 39 5.1 Lens Shape and Function  40 5.2 Convex Lenses  41 5.3 Concave Lens  41 5.4 Lens Coating  42 5.5 Anti-reflective Coating  42 5.6 Prisms  43 5.7 Magnification  44 5.8 Numerical Aperture  44

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5.9 Resolution  46 5.10 Focus  46 5.11 Aberrations  47 References 47 6 The Anatomy of the Microscope 49 6.1 Styles of Microscope  50 6.2 The Upright Microscope  50 6.3 The Inverted Microscope  52 6.4 The Stereomicroscope  53 6.4.1 Types of Stereomicroscope  54 6.5 Petrographic Microscope  54 7 The Eyepiece 57 7.1 Compensating Eyepieces  58 7.2 Eyepoint  58 7.3 Interpupillary Distance  59 7.4 Using Eyepieces as a Glasses Wearer  59 7.5 Diopter Eyepieces  60 7.5.1 How to Adjust a Diopter Eyepiece  60 7.6 Reticle  61 7.6.1 How to Calibrate a Reticle  62 7.7 Cleaning an Eyepiece  62 7.8 Scale Bars on Digital Systems  63 References 63 8 The Objective 65 8.1 How to Read an Objective  67 8.1.1 Achromatic and Apochromatic  67 8.1.2 Infinity Correction  68 8.1.3 Magnification  68 8.1.4 Numerical Aperture (NA)  68 8.1.5 Planar  68 8.1.6 Refractive Index (RI)  70 8.1.7 Thread Depth and Brand Compatibility  70 8.1.8 Working Distance (WD)  70 8.2 Air Objective  71 8.3 Oil Immersion Objective  72

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8.3.1 How to Use an Air Objective  72 8.3.2 How to Use an Oil Immersion Objective  74 8.4 Cleaning an Objective  77 8.4.1 Cleaning an Air Objective  78 8.4.2 Cleaning an Oil Objective  78 8.5 Summary of Objective Codes  79 Reference 79 9 The Stage 81 9.1 Focusing the Sample   83 9.2 Slide Navigation Controls  83 9.3 Petrographic Microscope Stage  84 9.4 Stages for Inverted Microscopes  85 10 The Condenser and Diaphragm 87 10.1 The Diaphragm  88 10.1.1 The Disc Diaphragm  89 10.1.2 The Iris Diaphragm  89 10.1.3 How to Centre the Iris Diaphragm  91 10.2 The Condenser  92 10.2.1 How to Focus the Condenser  93 References 94 11 Light Sources 95 11.1 Different Types of Light Source  96 11.1.1 Internal Light Source  96 11.1.2 External Light Source  97 11.2 Different Types of Bulb  97 11.2.1 LED (Light-Emitting Diodes)  97 11.2.2 Incandescent Bulbs  99 11.2.3 Halogen 100 11.2.4 Mercury 101 11.2.5 Metal Halide 101 11.2.6 Fluorescent 101 11.3 White Balance 102 References103

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12 Choosing the Right Technique105 12.1 Brightfield 107 12.1.1 What Does It Look Like? 107 12.1.2 When to Choose Brightfield 107 12.1.3 Tech Specs 108 12.1.4 Sample Requirements 108 12.2 Darkfield 108 12.2.1 What Does It Look Like? 108 12.2.2 When to Choose Darkfield 109 12.2.3 Tech Specs 109 12.2.4 Sample Requirements 110 12.3 Phase Contrast 110 12.3.1 What Does It Look Like? 110 12.3.2 When to Choose Phase Contrast 110 12.3.3 Tech Specs 111 12.3.4 Sample Requirements 111 12.4 Z-Stacking 112 12.4.1 What Does It Look Like? 112 12.4.2 When to Choose Z-Stacking 112 12.4.3 Tech Specs 112 12.4.4 Sample Requirements 113 12.5 Stereomicroscopy 113 12.5.1 What Does It Look Like? 113 12.5.2 When to Choose Stereomicroscopy 113 12.5.3 Tech Specs 113 12.5.4 Sample Requirements 113 13 Sample Prep115 13.1 Prepared Slide Sets 116 13.2 Equipment for Preparing Samples 116 13.2.1 Glass Slides 116 13.2.2 Coverslips 118 13.2.3 Stains 119 13.2.4 Dry Mount/Wet Mount and Temporary/ Permanent Mount 119 13.3 Health and Safety 120 13.4 Simple Thin-Prep Method for Brightfield Microscopy 120 13.4.1 Cutting the Sample 121 13.4.2 Fixation 122 13.4.3 Staining 123 13.4.4 Coverslipping 123

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14 Image Capture127 14.1 Mounted Camera 128 14.1.1 C-Mounted Cameras 128 14.1.2 Modular Camera 129 14.1.3 Software 130 14.2 Eyepiece Camera 130 14.2.1 Software 131 14.3 Camera Phone Adapter Mount 131 14.3.1 Software 131 14.4 C-Mount Adaptor and a Regular Camera 131 14.5 Imaging Software 132 14.6 Good Practice Points for Image Capture 132 14.7 What Is a Digital Image? 133 14.7.1 How Is a Digital Image Created? 133 14.7.2 What Is a CCD? 133 14.7.3 Pixels 134 14.7.4 Resolution in Digital Images 134 14.7.5 Greyscale 135 14.7.6 RGB Colour 135 14.7.7 Bit Depth 135 14.7.8 Compression 136 14.7.9 Image Formats 137 15 Troubleshooting139 15.1 A Word of Warning 140 15.2 Problem: There Is No Light Visible in the Eyepieces, or the Light Is Dull or Uneven 141 15.2.1 Power 141 15.2.2 Light Source 141 15.2.3 Diaphragm and Condensers 142 15.2.4 Stage 144 15.2.5 Objective 144 15.2.6 Shutter 145 15.2.7 Eyepieces 146 15.2.8 Camera 146 15.3 Problem: There Is a Shadow in the Image 148 15.4 Problem: Cannot Focus During Air Imaging 148 15.5 Problem: Issues During Oil Imaging 149

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15.6 Problem: The Light Is Coming Through the System, But It Is Uneven 15.7 Choosing the Right Light Level 15.8 Colour of the Sample Is Incorrect 15.9 The Wring Test 15.10 Centring an Objective on a Petrological Microscope 15.11 How to Change the Bulb in an External Light Source

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Glossary155

About the Author

Dee Lawlor  is a science writer and microscoper from Dublin, Ireland. She achieved her degree in zoology from the University of Aberdeen in 2010 and then pursued a master’s in microscopy and image analysis from the University College Dublin in 2012. After graduation, Dee worked in microscopy sales and then spent several years working as a technical support engineer for digital imaging systems for Leica Biosystems. Dee has a great passion for microscopy—both academically and in application. In her spare time, she collects and renovates antique microscopes and telescopes. Her writing career started in 2016 when she started writing articles for a range of different scientific websites. Her specialist topics are microscopy, biology, and environmental sciences. She launched her own microscopy blog ‘What the Microscope Saw’ in November 2017, where she regularly publishes special interest articles. Her work is aimed at a wide variety of audiences, and her articles are written for the enjoyment of everyone—scientists and nonscientists alike. After several years of problem-solving for scientists all over the world she has decided to put pen to paper, to share her knowledge and passion for microscopy in her first book Introduction to light microscopy—tips and tricks for beginners.

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1 Introduction

The Microscope not only assists studies, and develops objects of profound interest, but it also opens up innumerable sources of entertainment and amusement, in the ordinary conventional acceptance of these terms; -it discloses to us peculiarities and attractions in abundance; -it impresses us with the wonderful and beautifullyskilful adaptation of all parts of creation, and fills our minds with additional reverence and admiration for the beneficent and Almighty Creator Jabez Hogg [1], The Microscope.

Abstract  The discoveries, advancements, and technologies that we enjoy today are the cumulative effort of millions of curious people over thousands of years. From the people who invented the wheel, the Greek philosophers, Renaissance explorers, instigators of the eighteenth-century enlightenment, Victorian taxonomists … as a contemporary scientist, you are the latest link in a very long chain. The history of science—and how we have arrived where we are today—is a fascinating topic. I have started this book with a brief history of the microscope. I have also included a brief history of glass, as this has had a direct impact on the development of microscopy. While the history of science is not essential knowledge, I encourage all scientists to learn about the evolution of their field and to explore the path that has brought us to where we are today. Nothing is a greater symbol of science and discovery than the microscope. Microscopes are an easily accessible technology and they have the power and potential to show us the world from a whole new point of view. Edward Tufte © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_1

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said, ‘the commonality between science and art is in trying to see profoundly— to develop strategies of seeing and showing’. The microscope has been, and continues to be, a huge contributor to education and our understanding of the world.

1.1 History Optics—the study of light—is one of the oldest branches of science. The word ‘optics’ was coined by the Greeks and comes from the Latin ‘ta optika’ which means ‘look’ or ‘appearance’. Light has been a source of great fascination for many of the great minds throuhgout history. In 300 BC, Euclid wrote one of the earliest known works on optics, in which he described his theories on the geometry of vision. In 350 BC Aristotle wrote in Problemata about the magnification of light and images. In 1021, Ibn al-Haytham, the Arab mathematician, astronomer and physicist, wrote the Kitāb al-Manāẓir (the Book of Optics). Many of the theories and ideas suggested by these great minds have today been proven to be correct. Interestingly, any the mistakes were mostly made on the biology side, but much of the physics was spot on. The history of the microscope stretches back to 750 BC Assyria, where we find the earliest known examples of manufactured lenses. The Egyptians and Mesopotamians are credited with inventing glass; however, many cultures developed the technology independently for themselves. The Chinese, for example, are known to have been making glass from before 200 BC [2]. The lenses are the most important part of the microscope. Their quality, purity, precision, and alignment are essential for creating a high-quality image. The oldest known lens in the world is the Nimrud lens, which currently resides in the British Museum. Also known as the Layard lens, it is a 1.5 inch diameter piece of rock crystal, originating from modern-day Iraq. Research on the lens suggests that it has a focal length of 12 cm and a magnification of approximately 3×. There are several theories as to its original use—it was possibly used as a magnifying glass, used for concentrating sun light to start fire, or possibly even part of an ancient telescope, but it could also just have been for decorative purposes. It is made from rock crystal and thus the quality of the lens has not deteriorated over time. Early glass was full of impurities and imperfections and lenses made from crystal were considered to be superior for much of history. Quartz crystal was a popular choice as it is renowned for its clarity and strength, which makes it ideal for polishing and grinding. In fact, quartz crystal is still used to make the prisms in many microscopes manufactured today, and crystal is known to not degrade the way glass can over

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time. Around 100 AD, Egyptians glassworkers in Alexandria made a significant leap in glassmaking technology. They made  improvements in furnace technology and this allowed them to reach higher temperatures and to have better control over the heat. This improved the quality of the glass by ensuring a more even and thorough melting and mixing of the components. They added manganese oxide to the glass mixture and created  the first clear  glass.  The knowledge and skills travelled west into Europe and eventually we would see every country develop it’s own trade and techniques, experimenting with different temperatures and chemical compositions. Glassmaking occurred all over Europe but it was  Venice that became  the glassmaking capital of the Western world. The world famous Murano glass was first produced in Venice in the thirteenth century and they would also become the finest mirror-makers in the world—in fact, it was Venetians who made the mirrors for the Hall of Mirrors in the Palace of Versailles (they worked in France though). Italy is credited with inventing the first  reading glasses in the thirteenth century. Large lenses with a gentle curvature give a low level of magnification. This low level of magnification was found to allow a person to read a book more comfortably or to see an object in the distance with better clarity. Over time, different lenses were made to correct different visual conditions such as short-sightedness, long-sightedness, and astigmatism (when the shape of the eyeball is more ovoid than spherical). The magnifying glass was a common instrument by the sixteenth century and was used by many naturalists to examine samples. The large, gently curving lens of the magnifying glass were of low to medium strength magnification. For higher levels of magnification, the loupe was used—the smaller lens, with a stronger curve, gave a higher level of magnification. The loupe is best known as the instrument used by jewellers to examine stones and settings.  The invention of the first microscope is credited to Hans and Zacharias Jansen in the late sixteenth century. The Jansens were a Dutch father and son duo who worked as opticians. Using their extensive knowledge of lenses, the Jansens found that when they aligned several lenses in a tube, they could view objects at much greater magnifications than what could be achieved using the single lens of a magnifying glass or loupe. Their invention looked like a small telescope and it was the first compound microscope. In the seventeenth century, the Flea Glass was developed. The Flea Glass was composed of two lenses—one convex and one flat—that were mounted in a circular frame, on a wood or ivory handle. It looked very much like a small magnifying glass and was used in much the same way—the user would hold it close to the eye and use the ambient light for illumination. The Flea Glass differs to the magnifying glass in that the lenses were smaller. The Flea Glass

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was available in a range of magnifications, but was mostly low to medium magnification.  Leeuwenhoek’s microscope—also invented in the seventeenth  century— was an advancement on the Flea Glass. It was a simple design where the sample was placed on the point of a pin. The pin was attached to a small board, with a small and highly curved lens set into it. The user held the lens up to their eye while facing a light source and the sample was focused by moving the pin nearer to, or further away from, the lens. Leeuwenhoek himself described how samples looked best when viewed at moderate magnification, as this gave the best balance of light and resolution. Technically, the Leeuwenhoek should be classed as a loupe, as it is a small, single, medium magnification lens. To posterity, however, it is considered a microscope. Throughout the seventeenth century, the microscope saw some of the greatest scientific discoveries come to light. A well-known example to biologists all around the world is Robert Hookes ground-breaking book, Micrographia. It was first published in 1665 and was one of the world’s first public introductions to the cell and other microscopic wonders. It quickly became a best seller and is still in publication today. Hooke is also believed to have been the first microscoper to accurately calculate magnification. By using a glass scale which had been accurately divided into portions of an inch and then seeing how many sections were visible under different objective lenses, he concluded that ‘for if one division, as seen with one eye through the microscope, extends to thirty divisions on the rule, which is seen by the naked eye, it is evident that the diameter of the object is increased or magnified thirty times’. An interesting fact about Hooke is that he was acutely aware that he was limited by the technology of his time, and in Micrographia he admits ‘so ill and imperfect are our Microscopes’. Hooke’s microscope was a tube model microscope, typical of the seventeenth  century contemporary models. This style of microscope was more along the lines of the design developed by the Jansens (a tube with multiple lenses). As the name would suggest, the microscope constitutes a wooden or paper tube, with lenses at the bottom end and an eye cup at the top. In the latter eighteenth century, they would start to be made of brass. There were several interchangeable lenses of different magnifications and these were the first objectives as we would recognise them today. The light was provided either by a mirror reflecting the ambient light or by an oil lamp, whose light was focused through a water-filled glass globe onto the sample. The microscope did not escape the elaborate aesthetic of the eighteenth century and we see these machines become miniature works of art. The Baroque style in the first half of the eighteenth century and then the Rococo style in the latter half, were  applied liberally to microscopes. Tubular style

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microscopes in the later eighteenth century stood on three legs, which made them sensitive to vibrations. The wood and paper bodies were also subject to warping. A significant advancement in microscopy that was  made  in the 1730’s was the development of the achromatic lens. Chromatic aberrations result in the different colours of light being spilt and a rainbow effect being seen in the image. Charles Hall discovered that by adding an extra lens, he could realign the colours of light and thereby resolve the issue of chromatic aberrations. See Sect. 5.11 for more on chromatic aberrations and how they are resolved. Similar to the Leeuwenhoek model, in 1740 the German physician Johann Lieberkuhn developed a model which was a lens positioned in the centre of a silvered speculum, and this helped add more light for the user. Lieberkuhn would go on to create several different styles of microscope, including the solar microscope which debuted in 1738. The solar microscope was similar in function the camera obscura, as it could be used to project the image of the sample onto a wall, thereby displaying it for all to see. Throughout much of the sixteenth, seventeenth and eighteenth century, the microscope was seen as a novelty item. Of course, it was also being used to make scientific discoveries, but the technology was still limited. Achievements such as Robert Hookes Micrographia are a beautiful example of what the imaging technology at the time could achieve, but we still did not have the magnification, clarity, and resolution to delve inside the cell. It was in the nineteenth century when the industrial era arrived and work left the home in favour of the factory, office, or lab, that the microscope became a real working machine. The microscope as a toy has endured, but the designs for work and the designs for play, diverged significantly during the industrial revolution. To meet its new role as a working machine, the microscope became sturdier and more robust. Microscopes were now made out of brass or cast iron. Decoration was done away with and the characteristic U-shaped base came along. The heavy base helped resolve the vibration issues that the eighteenth  century tripod suffered. The heavy base also shifted everything upwards—the light source could now sit directly underneath the stage, as opposed to being shone in from the side. In 1830, Joseph Lister resolved the issue of the spherical aberration by optimising the distance between lenses. See Sect. 5.11 for more on spherical aberrations. The new reclining model of microscope (known as short-stand, as opposed to the eighteenth century high-stand) allowed for the user to sit instead of needing to stand while viewing the sample, further improving stability of the microscope and the image. Throughout the nineteenth century, mass production of microscope began. Microscopes were being produced all around Europe—with England, France, and Germany being the main contributors. European microscopes were

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openly traded all around Europe and exported to North America. North America did, of course, start manufacturing its own range of microscopes, which were reported as being enormously superior to the European models [3]. In 1859, it is reported that approximately 2000 large microscopes were manufactured in Europe in a year; by 1869 it was closer to 4000. This brought down the price which made them more accessible for scientists and non-­ scientists alike. Up until the late nineteenth and into the early twentieth century, the illumination of the sample was done either with focused lamp light or with light reflected by a mirror. Poor lighting had been a limiting factor in the quality of the image and the next leap in image quality came with the addition of the electric light. This gave us brighter illumination, which increased the sharpness and ­resolution of the image. By seeing brighter, we could see more detail and at higher magnifications. Today the microscope has become a highly sophisticated piece of machinery. Advancements in optics, lighting, and newer fields such as digital imaging are constantly improving and refining the technology. At its core, however, it is still just a tube with lenses. I don’t say this to be disparaging, but to be reassuring to the user that no matter what system they are faced with, a strong knowledge of the basics will get them through. The future of microscopy is in the ability to see smaller and clearer. We have already imaged a single atom and scientists are continually breaking the barriers of what we thought was possible. In 2014, scientists brought the optical microscope into the world of nanoscopy—achieving a resolution less than half the wavelength of light. This has allowed them to visualise individual molecules, such proteins moving in inside a living cell. Given these advancements and how quickly technology is moving, one can only imagine what we will be able to see in the next 10 or 20 years. The human species has achieved incredible things over the last 500  years, and the microscope has been there to watch.

1.2 The Importance of Glass in Microscopy For glass to be suitable for optics, it must be of the highest quality. Early optical lenses were made from flint glass—which is a variety of highly pure lead glass that was developed in 1676 in England by adding lead oxide to the glass mixture. Any artefacts or flaws in the glass can cause refraction issues, which will result in loss of image quality.

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Defects that occur during the manufacturing process include bubbles, discolouration, striations, and particulate matter. These issues occur for a number of reasons, but mostly from insufficient melting of the glass mixture, or contamination of the glass mix. Glass that is used for optics must be very pure and free from these artefacts. The first sources of directed light in microscopy were mirrors, and mirrors have played a huge role in the evolution and development of microscopes. The Phoenicians were the first to create glass mirrors. Previously, highly polished sheets of metal such as copper or silver were used for mirrors. The Phoenicians used glass backed with tin, but since the glass itself was not even in thickness across the sheet, the image was not particularly good. Glass mirrors did not outshine their metal sheet counterparts until the thirteenth century, when flattened glass coated with lead antimony was manufactured in Germany. Mirrors were initially used for directing light into the light path, but today they are mostly used for splitting and redirecting light through the imaging system. 

1.3 What Classifies as a Microscope? The simplest questions are often the ones that are the most overlooked. What actually classifies as a microscope? When does a magnifying glass become a loupe, and when does a loupe become a microscope? The answer is the lenses. The number, size, shape, arrangement, and power of the lenses define the machine. • A magnifying glass is composed of a single, large lens that has a gentle to moderate curvature. The lens can be planoconvex but is normally biconvex. The magnification of a magnifying glass is low to medium. • A loupe is a single, small lens that has a more prominent curve. The lens is biconvex and the magnification is medium to high. • A microscope is composed of a tube with two or more lenses. The magnification of a microscope ranges from low to high. The characteristics and behaviour of lenses are discussed in greater detail in Chap. 5.

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1.4 Why We Find Microscopes Appealing Humans are visual animals and large areas of our brain are dedicated to processing visual information. Our ancestors were visual hunters—using our forward-facing eyes to judge the distance of prey and to take aim at our targets. A large amount of our communication is done through our eyes, and we often judge a person’s character or intentions by the appearance or movement of their eyes. Humans are one of the only species that has a white sclera (the ‘whites’ of our eyes). This is a communication aid, as the white of our eyes allows us to give directional information with a slight glance. Interestingly, dogs have a white sclera too in order to communicate more efficiently with us—a marvellous example of evolution. Socially and culturally, we put huge weight into vision—‘I saw it with my own two eyes’, ‘Look at me when I’m talking to you!’. They are an indicator of our health, feelings, and intentions. It is understandable that the microscope—something that allows us to see the microscopic world for ourselves, with our ‘own two eyes’—would have such a huge impact on our curiosity and understanding of the world. Traditional microscopes are light microscopes—they use visible light to create the image, and this will be discussed in further detail in later chapters. The technology has expanded to use different sources of information for image creation. Atomic force microscopy (AFM), for example, uses touch to image the sample, creating a topography-style image. Electron microscopy (EM) uses electrons to visualise the sample and is used to create images with a resolution of microns! The technology is ever expanding, and we are continually refining and improving the quality and utility of imaging. So why learn, and read, and write about microscopes? Firstly, because microscopy is a skill. Every area of science uses microscopes and they have found their way through many other different industries. Engineers use microscopes to inspect parts for microfractures and wear and tear. The second reason is purely because everything is more interesting under a microscope! The human urge to learn and know always gets the better of us, and we cannot resist looking at the microworld. Microscopes have seen humanity through some of the greatest discoveries science has made—the cell, bacteria, and DNA. All the things that were hiding in plain sight are brought into the light by the microscope.

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References 1. Hogg J (1861) The microscope: it’s history, construction & application. Routledge, Warne, and Routledge, London 2. Fuxi G et  al (2006) Origin of Chinese ancient glasses  – study on the earliest Chinese ancient glasses. Sci China Ser E Technol Sci 49(6):701–713 3. Hagen H (1869) My experience in the use of various microscope. Mon Microsc J 1869:321–325

2 The Advantages of Learning Traditional Microscopy

Science makes people reach selflessly for truth and objectivity; it teaches people to accept reality, with wonder and admiration, not to mention the deep awe and joy that the natural order of things brings to the true scientist Lise Meitner

Abstract  Microscopy is a highly advanced technology, and every year there are new and exciting capabilities being developed. It can be tempting to skip the obstacle course and head straight for the finish line, but we experience and learn very little by doing that. The essence of knowledge is understanding. Understanding is achieved through experience. And experience takes time, trial, and error. To be a skilled microscoper, one must start from the beginning. Mastering the basics will make using more advanced systems much easier for you. I have seen scientists sitting in front of their multi-million euro imaging systems, complaining that it’s not working, only to find that they were putting the slide upside down. Invest in your basics, it will pay dividends.

Microscopy, like all areas of science and technology, is constantly moving forward. The compound microscope was invented in the late sixteenth century and since then it has gone through several iterations [1]. The basic idea and design, however, has largely remained the same. Using a series of lenses, light from the sample is manipulated to create an image that can be seen by the human eye. The technology and theories used in microscopes are the same as © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_2

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those used in telescopes and cameras. Each machine just has a slightly different arrangement and is packaged slightly differently. One of the most significant changes to microscopy (and all imaging) has been the digital camera. Digitalisation has made a huge impact in the world of imaging. By adding a digital camera to the microscope, the user is now able to view their samples on a computer monitor. Images can be captured, edited, analysed and stored indefinitely.  Never before have scientists been able to share information as easily as they can today. Digital imaging is now a well-­ developed technology and has become more widespread amongst working pathologists and researchers [2]. In an age of automation, machine learning, and incredibly advanced softwares, it is easy to understand how traditional microscopy can be overlooked. However, one should never underestimate the value of skill—and good ­microscopy is a skill! To understand (and appreciate) the advanced, we must start at the beginning, and there are many advantages to be found in going old school.

2.1 Skill Acquisition Microscopes are used in nearly every facet of science and also in many other industries such as engineering, quality control, computer chip manufacturing, and jewellery making—to name but a few. While most people can take a good guess at how to use a microscope, few people actually understand the workings and how to optimise the image. As noted in a nineteenth-century microscopy journal, “an experienced observer will often see much better with his own imperfect instrument…than another person would do with a far superior microscope” [3].  A user with good skills can get a great result from an average machine. An unskilled user will achieve very little, no matter how advanced the machine. By mastering the basic models and concepts, the user will find advanced systems (fluorescence, confocal, electron microscopy etc.) much easier to use and understand. 

2.2 Enjoying Knowledge The optics involved in microscopy are an interesting topic in themselves and the concepts involved are not particularly difficult to learn. However, many microscope users have no knowledge of optics and how the consitituent parts of their microscope affect the behaviour of light. By learning the basics of

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optics and applicable concepts such as refraction and how to follow the light path, the user will be well equipped to deal with a range of imaging issues. For example, if the image will not focus, the user can call upon their knowledge of refraction and focal points to surmise that the distances between lenses (or between the lens and the sample) is not correct. Learning should not be limited to a single field of expertise. As a biologist, I have found many benefits to learning some physics. Likewise, I know physicists who have benefitted from learning some biology. It has only been in the last century that scientists have become pigeon-holed into specific specialities. Previous to the nineteenth century, when science became an industry, a person of science was known as a natural philosopher. They would study a broad range of topics including biology, physics, geology, chemistry, astronomy and engineering. Do not limit yourself—a broad knowledge and an open mind are great assets.

2.3 Budget-Friendly In labs where the budget might be tight, a traditional microscope is much more affordable than a digital system. Second-hand microscopes (once properly cared for) will function just as well as a new ones. The parts of a traditional microscope can really only fail if they suffer some kind  of physical damage—such as a cracked lens or fluid damage. Parts for a manual microscope are usually cheaper to replace than parts for a digital  system. The user can often use parts from different  machines and second-hand parts are widely available. If a part on a traditional microscope needs to be replaced, this can often be done by the user. Replacing a part in a digital system will usually require a specially trained engineer, which can be expensive.  If images need to be captured, it is quite achievable to purchase a digital camera that can be attached to a traditional microscope. Most microscopes can be fitted with a standard sized C-mount that will allow a camera to be attached to the system. There are even attachments now that can connect the microscopes eyepiece to a camera phone. See Chap. 14 for more information on cameras.

2.4 Low Training Requirement Microscopes are an interesting technology in that people tend to have an intuition for how to use them, having seen them on TV or in movies. It is common to see at science events, people who have never studied science will

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pick up a slide, place it on the stage and look down the eyepiece without any hesitation. This means that training people to use a traditional  microscope requires only a small amount of time and effort. Digital systems tend to require organised training with end users to ensure that  they are using the system correctly. This also requires the user to have a pre-existing knowledge of how to use a computer. While this last point may sound silly, I have met many highly-experienced pathologists who are computer-phobes and refuse to use digital systems. Being classically trained, they want to see the sample with their own eyes. 

2.5 Robust A good quality microscope is quite a robust piece of machinery and is often designed to minimise the potential for damage. They are heavy and solid, which reduces vibrations and means they are difficult to knock over. The dials for adjusting focus and for swapping objectives are turned by hand. This reduces the force being applied to the moving parts and also introduces a certain degree of consciousness in the user. The range of movement of the moving parts is also limited, so the risk of overexerting the parts is reduced. The vulnerable parts of the microscope are the lenses (including the condenser) and the diaphragm. The diaphragm is made of overlapping leaves and these can come loose and become misaligned. They can, however, usually be fixed with a little careful handling. See Chap. 10 for more on the diaphragm. Short of crashing the objective through the slide, there is very little damage an inexperienced microscope user can do to a traditional microscope.

2.6 Reliable Technology based on mechanics tends to be more reliable than technology based on electronics. The parts are usually more robust and less prone to breakage or malfunctions. Traditional microscopes are controlled by the hands of the user: inserting and removing filters, adjusting diaphragms, focusing, etc. Human intervention and intuition can avert and resolve mistakes quite easily. Digital systems move their parts using electronics and software, which can be automated or controlled by the user. If the electronics fail or malfunction, they cannot be so easily fixed or intervened upon, and imaging will cease until the issue can be fixed by a trained engineer. In digital systems, the potential for electrical and

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computer-based faults is introduced. The parts for a traditional microscope do not require power or software to adjust them, and therefore they are not prone to failure.

2.7 Easy Troubleshooting Troubleshooting an imaging issue can be a daunting task to the inexperienced user. This is where the user’s knowledge of the basic technology will become a great advantage. On a traditional microscope,  the user can see all the ­constituent parts and easily move and adjust parts by hand while looking through the eyepiece. This makes troubleshooting quick and intuitive for those who understand how the microscope works. Troubleshooting on digital systems often involves knowledge of computers and software.

2.8 The Technology Doesn’t Age A well-built microscope, in good condition, will always function as a microscope. I have microscopes in my collection that are over a hundred years old and they can still be used to look at a prepared slide. In contemporary imaging, users are used to images that look a certain way—mostly, they are used to very bright images. A sample illuminated with a mirror seems incredibly dull to the modern user, so naturally, it will not look as good. The lighting does affect the image quality, as discussed in Chap. 11, but I have been able to achieve a perfectly clean image by shining the light on my phone up through an old microscope. Once the lenses are intact, a microscope will do what a microscope does.

2.9 Easy to Move I’m a biologist by trade, and like many biologists I grew up dreaming of exploring far off places. It will come as no surprise that there are not many digital imaging systems that can be carried around the Amazon or the Sahara in a backpack. More and more, scientific and medical labs are moving to where they are needed. Doctors Without Borders, for example, provide front-­ line medical care in rural and developing areas, and areas of the world affected by war or natural disasters. Labs in these regions are often subject to unreliable power which means there are frequent power cuts [4]. A traditional micro-

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scope with a mirror can be used regardless of what power is available. Contemporary brightfield models can be run on batteries and even on solar power. Advanced digital systems can have trouble recovering from power cuts, especially if they are connected to a computer. If a traditional microscope is on during a blackout, no major intervention is required to bring the system back to work, other than simply switching it back on (or directing the mirror to an alternative light source). Traditional microscopes can also be easily stored and transported  and usually do not require any special set-up or break-down.

References 1. Bradbury S (1967) The evolution of the microscope. Permagon, New York 2. Hedvat C (2010) Digital microscopy: past, present, and future. Arch Pathol Lab Med 134(11):1666–1670 3. Hagen H (1869) My experience in the use of various microscope. Mon Microsc J:321–325 4. JOURNAL, CISR (1999) Doctors without borders. J Mine Action 3(3):Article 21

3 Buying a Microscope

It is not many years since this invaluable instrument was regarded in the light of a costly toy; it is now the inseparable companion of the man of science Jabez Hogg (1861) ‘The Microscope’

Abstract  One of the wonderful things about microscopy is that there is something for everyone. Whether you are a hobbyist buying your first investment piece or a lab tech stocking up a lab for students, the options are many! However, so many options can be mind-boggling, especially if this is your first time buying a microscope. In this chapter, I have listed some questions for you to think about, so that you can figure out (or at least narrow down) your needs and wants. Second-hand microscopes can be just as good as new ones, so I have included a list of questions to ask if you are looking at buying a second-hand machine.

Purchasing a microscope can be both an exciting and intimidating experience, especially if the user does not have much technical knowledge. There are many specifications to cover and many different options to choose from. Microscopes can be a major investment, so it is important to make a wise and informed choice. Remember that many systems are adaptable—for example, a brightfield microscope can easily be adapted for Phase Contrast and for Darkfield imaging. The user should consider the long-term requirements of the microscope and not just the immediate need. For example, if the user intends to do image

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capture in the future, they might choose a trinocular head instead of a binocular head, in anticipation of the work they will be doing later.

3.1 Decide What Is Needed This is the first thing the user needs to decide. What does the microscope need to be able to do? What result does the user want? The needs of a hobbyist are different to those of a researcher, so these are important questions to spend some time on.

3.1.1 Who Is Going to Be Using the Microscope? When buying a piece of technology, the experience of the user will determine the complexity of the system that is chosen. A user that is just starting in microscopy should choose something that is straightforward to use. A complicated piece of machinery, while tempting (especially when fuelled by beginners enthusiasm), can be a burden to use and care for. This just takes away from the joy of using the microscope. A nice mid-range microscope that comes with three or four air objectives and a diaphragm is a great start for new users, and accessories can be added later. How many people are going to be using the microscope? An individual user who is the sole curator of their microscope can easily keep track of parts and will know the usage history of their machine. If the microscope is going to be used by many people, it is best to choose parts that are not detachable, or at least to limit the number of parts that can be adjusted or swapped out. This is not a reflection on the ability of the users, but more to do with accountability if something gets lost or damaged. The age of the user must also be considered. For advice on microscopes for young users, see ‘Microscopes for Children’ in Sect. 3.3. School-­aged students and university students have different requirements and also have differing levels of responsibility. School-aged children are usually quite keen to see their samples and this often leads to rough handling of the microscope. Models with a basic (or no) diaphragm and fixed eyepieces are best. This way the student need only be concerned with placing and focusing the sample. University students can be introduced to techniques such as oil imaging and Phase Contrast, but the model of microscope should be robust and with as few detachable parts as possible. University microscopes are taken in and out of cupboards several times a week, so the potential for lost parts is reduced by having everything fixed. A researcher or PhD student who is purchasing a

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system will need to spend some time consulting the primary literature and their colleagues to narrow down their needs.

3.1.2 W  hat Type of Samples Are the Users Working with? Different sample types have different imaging requirements. See Sect. 12.2.4, for guidance on the needs of the sample. This question must follow ‘who is using the microscope?’. If there are many different users, than all of their sample requirements must be accounted for. If the microscope is for hobby or general use, then a straightforward brightfield model, that can be adjusted for different techniques, is a good choice.

3.1.3 How Often Is the Microscope Going to Be Used? A microscope that is going to be a workhorse for a lab should be a good quality, robust model, preferably from a reputable dealer who will also provide maintenance and support. Is the system going to be used regularly throughout the day or only occasionally? If the system will be used regularly throughout the day, does it need to be switched on and off, or can it be left turned on? This is important to know when choosing the light source, as certain types of bulb do not respond well to being constantly switched on and off. See Chap. 11 for more on light sources. If a system is not going to be used regularly, perhaps it can be shared with another user and the cost split? 

3.1.4 Where Is the Microscope Going to Be Used? Is the microscope going to be used in a lab, in an office, or on the kitchen table? Is there is a power source within reach? The user will need a sturdy surface to put the microscope on and room for a chair for comfortable viewing. The user will also need space to comfortably rest their arms on either side of the microscope. Is there heavy people traffic? If there is, this could introduce vibrations which will affect the image quality. Like all technology, microscopes should be situated away from water and cables should be out of the way, so as not to be a trip hazard. The microscope should be used and stored in a clean and dry room, as dust and humidity can damage the optics.

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3.1.5 W  ill It Stay Set Up or Will It Need to Be Packed Up After Each Use? If the microscope is going to be stored between uses, it needs to be easy to move. A built in light source makes taking the microscope in and out easier. External light sources introduce the issue of extra boxes and cables to manage, although the systems that require external light sources are usually lab machines. If the microscope needs to be stored, is there a dry, clean, dust-free place to store it? If the system is going to be permanently left out, does it have a dedicated power supply or will people be plugging and unplugging it? 

3.1.6 Does the User Require Image Capture? Does the budget include the extra cost of a camera? If a camera is being purchased, the microscope will need a trinocular head (which can also add extra to the cost). A new computer might need to be considered, depending on the software required. See Chap. 14 for guidance on choosing a camera.

3.2 Budget and Where to Invest Budget is usually the limiting factor when purchasing any piece of equipment. The budget that is available and the budget that is desired can often be two very different things. Luckily, the cost of microscopes is wide ranging. Second-hand microscopes can be just as good as new ones, so do explore the second-hand market. Reviewing a second-hand machine will be discussed below in Sect. 3.4. The optical components (the objective, eyepieces, and condenser) and the light source will be the biggest influencers on the quality of the image obtained from the microscope. The parts that contain glass (the lenses) are usually the most expensive part of the microscope—they are definitely the most important! It is wise to invest in the best objectives, eyepieces, and condensers that the budget will allow. The next most important component is the light source. Spend some time exploring the available options, speak to different suppliers, and plan long-term. If you have to choose, invest in the objectives first.

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3.3 Microscopes for Children Science is now being widely taught in schools to young children and the microscope maintains its place as a popular toy. There are many different science kits available for children that contain a child-sized microscope and accessories for preparing samples. These sets are great in that they are specifically marketed to be attractive to children and are generally not too expensive. A great choice for children is a handheld digital microscope. These can be easily found online and are not very expensive. While these don’t look like a typical microscope (and are not as attractively marketed to children), the image they capture is excellent and they are very easy to use. The main advantage of choosing a digital microscope for children is that they are plugged into a computer monitor or tablet to view the image. Children are prone to having trouble using an eyepiece. On microscopes with one eyepiece, children can find using one eye uncomfortable (often having to keep one eye closed with their hand). On microscopes with binocular eyepieces, children can have trouble getting the distance between the eyepieces right. From the parent or educators point of view, it can be hard to know if the children  are actually seeing the sample properly. By using the digital microscope with the image on a monitor, the teacher or parent can be sure that the child is seeing the sample. Another advantage of the digital microscope is that features can be easily pointed out on the monitor—something that can’t easily be done with a traditional microscope. The view of the sample can also be shared with multiple users at the same time, so sharing does not become an issue. Images can also be captured onto the computer  and then shared or printed—children love being able to proudly display their findings on the wall.

3.4 Buying a Second-Hand Microscope Second-hand microscopes can be a great purchase. Once a microscope has been cared for, it will always function as a microscope. Universities and labs sometimes have sales of microscopes to make space for new machines, and there is a healthy market online for second-hand microscopes and parts. As with buying anything second-hand, it is best to see the microscope in person before purchasing. Below is a list of the checks to make when looking at a second-hand microscope.

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Part

Question

1. Body of the microscope

A. If the body is metal, is there any rust or corrosion? B. If it is plastic, are there any broken parts?

2. Light source

A. Are the electrics safe? Check for damage to cables and connectors B. If the electrics are safe, does the light source work? C. What type of light source is it?

3. Moving parts

4. Lenses

A small amount can be treated with metal paint, but it is better not to have any If any part of the body is broken, this will negatively affect the alignment and movement of the parts Replacing broken light sources and cables can be done by a professional. If it is an external light source, a new one can be purchased If yes, move to question C If no, it might just need a new bulb, or it might need to have the electrics replaced Where can the correct bulbs be bought? Is it the type of light source you are looking for? If not, can you change it to the light source that you want to use? The movement should be free and non-restricted in all directions. When the dial is let go, the stage/objective/ condenser should hold its position, without any drifting If the moving parts cannot hold their position, then there are issues with the threading. This will cause drift in the focus and will make imaging laborious

A. Use the dials to move the stage in every direction to its full extent. Does it move smoothly and easily? Does the stage hold its position when the dials are released? B. Use the dials to move the objective and condenser up and down, to their full extent. Do they move and do they hold their position when released? Check each eyepiece and every A. Turn on the light source objective. Look for any cracks or and look down the scratches. Look around the edges of eyepieces. If the light the field of view. Dirt can be source does not work, use cleaned, but breaks and scratches a torch or the light on a cannot be repaired. If there are mobile phone breaks in the lenses, check if new eyepieces or objectives can be purchased B. This is also an opportunity It should be easy to move between the objectives with one hand. Each to check the movement of objective should click into place and the objective head not move. No other part of the microscope body should move while the objective head is being turned (continued)

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Question

5. Filters

A. While looking down the eyepieces with the light on, check each filter

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Like with the lenses—dirt is ok, cracks are not. If there are some cracks, check if compatible filters can be purchased The diaphragm is an easily damaged part. If any of the leaves are loose, missing, or misaligned, this will negatively impact imaging While not impossible to fix, it can be difficult to do. If replacing the diaphragm, an exact match must be used

6. Diaphragm A. Remove the diaphragm from the microscope if possible. If not, do this check while looking down the eyepieces with the light on. Open and close the diaphragm. Is the movement smooth? Do all the leaves open and close evenly. When closed, is the opening in the diaphragm perfectly centred? (see Sect. 15.2.3) 7. Condenser The movement and stability of the condenser was checked in step 3 The light should remain even across With the light source the field of view. As the condenser switched on, open the moves, the light levels and field of diaphragm fully. Look view will change, but it is the quality down the eyepieces and of the light that is important. There move the condenser up should not be any dark spots and the and down light should stay centred

4 The Science of Light

Learn to see. Realize that everything connects to everything else Leonardo da Vinci

Abstract  Microscopy is synonymous with biology and—in general—biologists won’t venture too far into the world of physics. I first learned about the physics of light during my masters. At first, it was terrifying because biologists and physicists speak different scientific languages. However, I have found it to be of great benefit to my microscopy, and I highly recommend that the microscoper spend some time learning about the physics and behaviour of light. Not only is it a very interesting and historic field of science, it will greatly help you in your understanding of imaging. You do not need to know equations or be able to do complicated mathematics to enjoy some physics. In this chapter, we shall take a look at some of the behaviours of light that are the most pertinent to microscopy.

Optics is the branch of physics that is dedicated to the study of light and how it interacts with matter. For the purpose of understanding microscopy, the user does not need to delve deeply into mathematical equations or complex diagrams to enjoy learning some physics. Simply by understanding some of the underlying behaviours of light, the user’s understanding of the microscope is enhanced. This knowledge will assist the user in troubleshooting and give them an understanding as to why they are making certain adjustments during imaging. © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_4

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In this chapter we will discuss some of the characteristics and behaviours of light that are relevant to microscopy.  We will then discuss photosensitivity and how we perceive light—how the eye works and how the brain allows us to ‘see’. Many of the parts and functions of the eye are analogous to the parts and functions of a microscope. It is an interesting comparison to make! The lens of the eye, for example, refracts light to focus it in the same way the lens in the microscope does. The iris of the eye adjusts the size of the aperture (the pupil) to control the intensity of the light being allowed in, the same way as the diaphragm on the microscope opens and closes to control the amount of light getting through. The retina is akin to the CCD in a digital system, and the brain is the computer that assembles the information and presents the image.

4.1 What Is Light? Light is a form of energy called electromagnetic radiation (EMR). EMR includes gamma radiation, x-rays, ultraviolet light, visible light, infrared light, microwaves, and radio waves.  EMR is released by the excited electrons of atoms. When an atom gains energy, the electrons become ‘excited’ (energised) and the excited electrons moves to a higher energy state in the atom (imagine them running up a stairs in excitement). The electron spends energy as it moves upwards, as it is moving against the gravity of the atom (the way running up a stairs is tiring). When the energy is used up, the electron starts to relax and return to its original  ground state. As the electron returns to its ground state (comes back down the stairs), an emission of energy occurs in the form of EMR. The type of EMR being emitted depends on the amount of energy being absorbed and released by the atom. For the microscopy techniques discussed in this book, the EMR we are interested in is visible light. The physics has been described in as straightforward a manner as possible  and equations have been kept to a minimum. An  in-depth knowledge of optical physics is not required for the level of microscopy in this book, but you might find yourself curious to know more.

4.2 The Basics of Light • The individual unit of light is the photon. • Photons can act individually or as a group. • When photons move in a group, they form a wave (Fig. 4.1).

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Light is measured and characterised by wavelength. Wavelength is defined as the distance between peaks of the wave (Fig. 4.2). Wavelength is denoted using the Greek letter lambda, λ (Fig. 4.2). If there is a large distance between the peaks, the wavelength is long, and this corresponds with relatively low amplitude (low energy) (Fig. 4.3). • If the peaks are close together, the wavelength is short, and this corresponds with having a higher amplitude (high energy) (Fig. 4.4). • Wavelength is measured in nanometres (nm). A nanometre is one billionth of a metre.

Fig. 4.1  An individual unit of light is a photon. Photons move together in waves

Fig. 4.2  Wavelength is measured as the distance between peaks in the wave

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Fig. 4.3  Long wavelength light has a low amplitude

Fig. 4.4  Short wavelength light has a high amplitude

4.3 Colours of Light The easiest way to define the visible spectrum of light is to think of a rainbow—red, orange, yellow, green, blue, indigo, and violet light. Note violet but not ultraviolet, and red but not infrared. • • • • • •

White light can be split into any of the individual colours of light. When all the colours of light are combined, white light is created. With the correct filters, any colour of light can be extracted from white light. The colour of light is defined by its wavelength. Each of the different colours of light has a characteristic wavelength. As the wavelength increases or decreases, the colour of light changes.

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4.4 Movement of Light There are several behaviours of light that are useful for the microscope user to understand—refraction, reflection, absorption, emission, and scattering. These behaviours all occur when light moves from one medium to another. In the vacuum of space, light can travel uninterrupted for millions of light-years (9.4607 × 1012 km)—this is the reason why we can see faraway stars. However, when light moves through a medium such as air or water, the light is interrupted by the particles of that medium. In the case of microscopy, an example of this will be light moving from the air into the glass of a lens and vice versa.

4.4.1 Refraction Refraction is derived from the Latin word refringere, meaning ‘to break up’. It is refraction that allows the microscope to magnify and focus the image of the sample. Therefore, it is the most important concept for the user to understand. Refraction occurs when light moves from one medium into another. In microscopy, the light mostly moves from air into glass and from glass back into air. In immersion (see Sect. 811 for more on immersion imaging), the light moves between fluid and glass. A certain amount of the light will also be reflected. Light that is being reflected can be considered to be losing information—in microscopy, we want the light to travel through the lenses, not to be reflected off them. To reduce the amount of light being reflected, the refractive index of the two media (in this case, the air and glass of the lens) should be as close as possible. Reflection is explained further below. • When light moves from one medium to another, it changes speed. • When the light changes speed upon changing medium, the light bends (Fig. 4.5). The density of the two media will determine how, and to what extent, the light is affected. For example, air has a low density and does not affect the light path too much, whereas water has a high density and scatters the light that enters it [1]. • The extent to which the light is bent is called the refractive index. • If a substance bends the light a large amount, the substance has a high refractive index.

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Fig. 4.5  The angle of the light changes when it moves from one medium into another

• If a substance bends the light a small amount, the substance has a low refractive index. • The refractive index (n) is calculated as the speed of light in a vacuum (c) divided by the phase velocity (v).  The equation for refraction is therefore: n = c / v A well-known example of refraction is the prism. White light enters the prism and as it travels through the crystal, it slows down and bends. The individual colours of light are separated and a rainbow is seen on the other side. See Sect. 5.6 for more on prisms.

4.4.2 Reflection Reflection is derived from the Latin prefix ‘reflex-’, meaning ‘to bend back’. Reflection is when light changes direction without being absorbed. The differ-

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Fig. 4.6  Light is reflected at the same angle that it strikes the reflective surface. A portion of light is reflected, and a portion is refracted

ence between reflection and refraction is that in refraction the light passed into the second medium (i.e. from the air into the water), but in reflection the light bounces off the second medium without passing through it. Reflected light is light that has been redirected off the surface of an object (Fig. 4.6). In microscopy, the user mostly  wants the light to be refracted, however, reflected light can also be advantageous. Darkfield microscopy, for example, uses reflected light. See Sect. 12.3 for more on darkfield microscopy. • Reflection occurs when light encounters a change in medium and the light is redirected instead of passing through. • When light is reflected, the angle of incidence (θi) is equal to the angle of reflection (θr). This means that the angle that the light approaches the medium at will be the same angle that it is reflected. Therefore, the equation to describe reflection is: θi = θr .

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4.4.3 Absorption and Emission When light strikes an object, the atoms the object is made of can absorb some of the energy of the light. When this happens, the intensity of the light reduces—a behaviour called the Beer–Lambert law of attenuation. The energy absorbed by the object can be converted into heat—this is why objects feel warm when left out in the sunlight. The energy absorbed from the photons can also be converted into an electrical signal, and this is how solar panels and CCDs work. • Light energy can be absorbed by an object and converted into another form of energy, such as heat or an electrical signal. This is known as absorption. Emission occurs when excited electrons in an atom return to the ground state. As the electrons are returning to the ground state, energy is released. There are two types of emission, spontaneous and stimulated. Spontaneous emission occurs when an atom goes from an excited (energised) state, to a relaxed (ground) state. Stimulated emission occurs when the sample is being ‘charged’ with a particular wavelength of light and then releases light. • Emission is when energy is being released from an atom and that energy can be released in the form of light. • Absorption and emission are essential behaviours for microscopy techniques such as fluorescent imaging. The sample absorbs light of one wavelength and then emits light of a higher wavelength (lower energy). This is a process called Stokes shift.

4.4.4 Scatter Light travels in a straight line in a vacuum because there are no particles to interrupt the movement of the photons. However, when the particles of light encounter other particles—such as the molecules in air—the photons will try to navigate through and around them. If the light has an energy that equals or exceeds the bond energy between the molecules, then the light can pass through relatively unchanged. However, if the molecules have a strong bond, for example, the molecules in water, the light particles are not able to continue on their path as easily and are scattered [2].

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• The scattering of light is the reason why the sky is blue. Light from the sun travels through space as white light. When it reaches Earth’s atmosphere, the molecules, dust, and moisture in the air cause resistance. Light at the blue end of the spectrum is scattered by the atmosphere and that is why the sky is blue. This is known as Rayleigh scattering or selective scattering, as only a particular colour of light is being affected [2]. In microscopy, we want to avoid scatter as much as possible, as this means our light information is being lost. 

4.5 Photosensitivity: How We Perceive Light Photosensitivity, the ability to sense light, is a characteristic shared by nearly every living organism on Earth. The sense of sight gives great advantages to survival. Many predators, including humans, hunt using vision—having two forward facing eyes gave us binocular vision, with which we can accurately judge distance. Prey animals use their broad peripheral vision to watch out for those predators. Birds and many species of fish use their vision to choose a mate based on colour and patterns. Plants detect light and actively grow towards it—a behaviour known as phototropism. Even bacteria are known to be responsive to light [3]. Photosensitivity does not just provide the ability to see. It controls the natural rhythm of our day and dictates our periods of activity and rest. This is known as the circadian rhythm and it is regulated by exposure to light. Changes in the duration of light exposure—including living in a world with artificial lighting—can change the circadian rhythms of many species, humans included [4].

4.5.1 The Evolution of Vision Photosensitivity evolved very early in life, and examples of early eyes have been found dating back to the lower Cambrian period, approximately 540  million years ago. The origin and early evolution of vision is hard to determine, as it is not well represented in the fossil record before the Cambrian period. However, the theory is that it started with organisms developing a patch of photosensitive cells on their surface. This patch of cells could detect simple changes in light, such as the shadow of a predator. These patches of cells then became invaginated. By adding an inward curve to the patch of photosensitive cells, directionality was achieved. Over time, this area closed over with a transparent membrane, which became the cornea. Fluid filled the

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cavity and eventually the lens evolved to focus the light [5]. The evolution of the quality of vision is believed to have started with simple shadow detection. Non-directional sensing led to directional sensing, and low-resolution vision led to high-resolution vision. Behaviour of the organism is believed to have influenced the evolution of vision and vice versa. As behaviour became more complicated, vision evolved to catch up. As vision became better, new more advanced behaviours could be developed [6].

4.5.2 Photosensitive Cells Photosensitive cells are a variety of cell that convert light into an electrical signal. This conversion is a process known as visual phototransduction. The photons enter the eye through the pupil and impinge on the photosensitive cells. These cells contain a protein called opsin. When opsin is excited by the energy of the photons, it initiates a chain reaction that converts the photon signal to an electrical signal. This electrical signal is sent down the optic nerve to the brain, and this is how vision is created [7]. There are two types of photoreceptor (light receiving) cells—rods and cones. These receptor cells line the inside of the eye and are mostly concentrated at the back of the eyeball in an area called the retina. The retina can be considered biologies version of the CCD (see Sect. 14.7.2 for an explanation of CCD). The fovea is a small area in the retina that is more commonly known as the blind spot, as this part of the eye cannot ‘see’. There are no photoreceptor cells in the fovea as this is where the optic nerve connects to the eye [7]. • Rod cells are incredibly sensitive and they are used to see in low light levels. This is known as scotopic vision. • Rods do not detect colour and this is why it is difficult to distinguish colours in low light conditions. • Cone cells require stronger light levels to trigger the nerve signal. They are used to see during the day, when light levels are relatively high. • Cones are used to detect colour. This is known as photopic vision. • Cones are better than rods at interpreting fine detail and movement [1].

4.5.3 Human Vision The human model of the eye is known as the simple eye. It is a fluid filled orb with an aperture (the pupil) at one side to allow the light in and a photosensi-

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tive surface (the retina) inside for the light to impinge on. Each eye has an optic nerve that connects it to the brain. Six muscles known as the extraocular muscles control the movement of the eye, and the brainstem controls pupillary action. The pupils will dilate and constrict to control the amount of light entering the eye, but also in response to what we are looking at. When something is interesting or appealing, the pupils open wider to capture more visual information. This is why our pupils dilate when we look at a person we find attractive or when we see something we really like or want. We are information gathering. Pupillary action is mostly involuntary, and most people cannot control it consciously. The brainstem is also responsible for a movement of the eye called saccades. When the eye is scanning an area, it does not move in a smooth motion, but in many small increments. This prevents our vision from blurring as our  eye moves [8].  A great way to demonstrate this is to watch the screen of a camera as you pan the camera around. When the camera is moving, the image blurs. Saccades prevents our eyes from doing this.

4.5.4

How We See

The job of the eye is to capture light information. It is the job of the brain to sort and organise this information into something useable. The light lands on the retina and it is converted into an electrical signal by the photosensitive cells. When an image lands on the retina, it is upside down and reversed left to right, because of the way the light is refracted through the lens of the eye. The brain however, will correct for this. The electrical signal is transmitted via the two optic nerves to the visual cortex at the back of the brain. Once the image information is constructed, it is interpreted by the brain. The brain is incredibly good at recognising shapes and patterns—this would have been a vital ability in our early evolution, when recognising predators quickly would have been a matter of life and death. This information then builds an image in the brain and you ‘see’.

4.5.5

Colour Vision

Humans are trichromats, which means that there are three different types of cone cells in our eyes. Each of the three is wired for capturing different wavelengths of light. Broadly, there is one type of cone cell for high-wavelength, one type for medium-wavelength, and one type for low-wavelength light, although there is some overlap between them.

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• The cone cells that are most sensitive to high-wavelength light are called L-cones. • L-cones are most sensitive to red light. • The cone cells that are most sensitive to medium-wavelength light are called M-cones. • M-cones are most sensitive to green light. • The cone cells that are most sensitive to low-wavelength light are called S-cones. • S-cones are most sensitive to blue light. It is the combination of these three types of cone cell, and their varying sensitivities, that allow us to see the different colours of visible light.

4.5.6

Variation of Vision Between Individuals

There is a philosophical question—how do I know that the colours I see are the same colours you see? Maybe each person sees something entirely different, but we all believe that our version of colour is the correct one? Variation amongst individuals is natural, and while the variation might not be as dramatic as each one of us seeing the world in an entirely different palette, there is variation between the vision of individuals that is pertinent to microscopy.

4.5.7

Colour-Blindness

Colour-blindness is a genetic condition where the affected person cannot see certain colours correctly  (or what we perceive as correctly). The genes that code for red and green vision are carried on the X chromosome. Red/green colour-blindness is significantly more prevalent in men, as they only have one X chromosome. If their X chromosome comes with a defective gene for red/ green vision, there is no matching gene on the Y chromosome that can compensate, resulting in red/green colour-blindness. Women have two X chromosomes, so if they have a defective gene on one, the other can compensate. It is a common misconception that women cannot be colour-blind. Although it is rare, women can be blue colour-blind, as the gene for blue cones cells is on a different chromosome that is not sex-linked [9]. In rare circumstances, women can possess an extra type of cone cell that can see ultraviolet light. This is known as tetrachromacy and is a genetic condition carried on the X chromosome.

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One of the great advantages of digital imaging is that it can compensate for individual perceptions of colour. As the colour of each pixel is determined by numerical values, if the user wants to be entirely certain that two pixels are indeed the same colour, they can simply consult the RGB values (see Sect. 14.7.6 for an explanation of RGB).

4.5.8

Variation in Physiology

Myopia (short-sightedness) and hyperopia (long-sightedness) are two very common conditions that affect an individual’s ability to focus. Thanks to modern optics, any individual with these conditions can resolve the issue quite easily with a pair of prescription glasses or contact lenses. This does, however, introduce a level of variation in the user’s imaging ability. When using a manual focus, a user who wears their glasses at the start of their imaging session, must keep wearing their glasses to ensure consistency in focus. A digital system—where the computer software is focusing the image—can compensate for this, but the glasses-wearing user must still be consistent when viewing and reviewing their images. The key is consistency.

References 1. Kawamura S, Tachibanaki S (2008) Rod and cone photoreceptors: molecular basis of the difference in their physiology. Comp Biochem Physiol A Mol Integr Physiol 150(4):369–377 2. Kerker M (1969) The scattering of light and other electromagnetic radiation. Academic Press, New York 3. Gomelsky M, Hoff WD (2011) Light helps bacteria make important lifestyle decisions. Trends Microbiol 19(9):441–448 4. Potter GD et al (2016) Circadian rhythm and sleep disruption: causes, metabolic consequences, and countermeasures. Endocr Rev 37(6):584–608 5. Lamb TD, Pugh EN, Collin SP (2008) The origin of the vertebrate eye. Evol Educ Outreach 1(4):415–426 6. Nilsson DE, Bok MJ (2017) Low-resolution vision – at the hub of eye evolution. Integr Comp Biol 57(5):1066–1070 7. Purves W et  al (2003) Life  – the science of biology. W H Freeman & Co, Gordonsville, VA ISBN-10: 0716719746, ISBN-13: 978-0716719748 8. Ibbotson M, Crowder N, Cloherty S, Price N, Mustari M (2008) Saccadic modulation of neural responses: possible roles in saccadic suppression, enhancement, and time compression. J Neurosci 28(43):10952–10960 9. Shulman R (2017) Color blindness and its illuminations. The Pharos, Autumn edition, pp 34–39

5 Introduction to Lenses

By the help of the microscopes, there is nothing so small, as to escape our inquiry Robert Hooke

Abstract  Like the saying ‘no foot, no horse’, one can say that ‘no lens, no microscope’. Your microscope is only as good as your lenses, and this is why it is important to understand how they work. Later in the book we will also discuss how to care for your lenses. Alongside a knowledge of light, a knowledge of lenses and how they work will solve most issues in microscopy. In this chapter, we will look at the characteristics of lenses and some of the key behaviours that are most relevant to microscopy. Optics is, of course, much more in depth than what is described here. But again, you do not need to learn equations or complicated concepts to have a workable understanding of your lenses.

The lenses are the essence of the microscope. They gather the light and provide the resolution, magnification, and focus needed to create the image. A microscope is composed of several lenses, prisms, and mirrors all placed at precise distances and angles from each other. Different types of imaging systems will have different combinations of these, but the overall recipe is the same. The quality of the glass in the lenses, and their precision of construction, will have a direct influence on the quality and capability of the imaging system. Before glassmaking technology refined its manufacturing processes, © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_5

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lenses were often made from ground and polished quartz crystal. Quartz was chosen for its purity, strength, and ability to transmit light evenly across the visible spectrum [1]. The crystals strength meant that it could withstand the grinding process required to shape it into lenses and prisms. Crystal lenses are also known to not degrade over time, the way glass lenses can [2]. The principles involved in lens making have been consistent throughout history— with the curvature and purity known to be the essential elements. Today, lenses are mostly made of glass. Before the industrial era, glass texture was uneven and often full of bubbles and impurities, making it unsuitable for lenses. However, improvements in the manufacturing techniques reduced the amount of impurities and imperfections in glass. Advances in optical engineering greatly reduced imperfections in the smoothness and ­consistency of the curvature and the surface of the lenses. This improved their refractive ability and accuracy, and the purer glass meant less photon scatter. These combined advancements have improved the optical qualities of glass lenses and have resulted in better image quality. The quality and precision of the lenses is the main reason for variation in the cost of objectives—expensive objectives use better quality glass and more precisely engineered lenses. Some microscope manufacturers even make their own glass, to ensure purity and quality. In this chapter, some of the characteristics of the lenses that are pertinent to microscopy will be discussed. An introductory knowledge of lenses, their behaviours, and how they work is immensely beneficial to the microscope user. With a fair understanding of lenses, the user will be better equipped for troubleshooting and for understanding why they must take certain actions during imaging. Please refer to Chap. 4 for more information on the behaviour of light.

5.1 Lens Shape and Function A microscope contains numerous lenses of different characteristic and curvatures. It is the combined behaviours of these lenses that creates the image for the user. An important characteristic of a lens is its ability to gather and transmit light. In microscopy, light is information, and the microscoper will want to capture as much light as possible. Good quality lenses are better at funnelling light, and therefore, less light information will be lost inside the system. Lenses are classified by their shape and the two main shapes are convex and concave. When predicting the behaviour of light refracting through a lens, the user should focus more on the lens surface that the light is exiting from.

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Fig. 5.1  Convex lenses bring the light rays together and focus the image

Of course, optics and the behaviour of light are much more complicated than what is described below. However, this introduction to lenses and the manipulation of light will give the user a great start.

5.2 Convex Lenses • A convex lens has a surface that curves outwards from the centre line (Fig. 5.1). • As the light exits a convex lens, the light rays are turned inwards (Fig. 5.1). • Convex lenses bring the light rays together and focus the image. • If the lens is convex on one side and flat on the other, it is known as plano-­ convex. If the lens is curved outward on both sides, then it is known as biconvex. An example of a biconvex lens is a magnifying glass.

5.3 Concave Lens • A concave lens has a surface that curves inwards towards the centre. • As light exits a concave lens, the light rays are turned outwards, and this enlarges the image. • Concave lenses spread the light rays apart and magnify the image.

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Fig. 5.2  Concave lenses bend the light outwards and magnify the image

• If the lens is concave on one side and flat on the other, it is known as planoconcave. If the lens is curved inwards on both sides, then it is known as biconcave (Fig. 5.2).

5.4 Lens Coating Many lenses are enhanced with special coatings that improve the optical qualities of the lens. These coatings are only a few micrometres (μm) thick and are used to fine-tune the optics of the lens. Users who wear glasses or who are interested in photography will be familiar with coatings such as scratch-resistant (which protects the lens from micro-­ scratches) or anti-reflective coatings (discussed below). Lens coatings can be recognised by a coloured tint on the lens surface when it  is viewed at an angle. As lens coatings are extremely thin, extra care is required when cleaning them. Special coatings also increase the price of an objective, so it is very important to take good care of them. See Sect. 8.14 for instructions on how to correctly and safely clean an objective.

5.5 Anti-reflective Coating When light encounters a medium, such as glass, two things happen. Some of the light passes through the glass (transmission) and is refracted and some is reflected away. This is a natural behaviour of light; see Sect. 4.5 and 4.6 for more about refraction and reflection.

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• Anti-reflective coating is used to reduce the amount of light reflected by the lens. This increases the amount of light that passes through the glass. • Anti-reflective coatings improve the efficiency of the light source by helping more of the light to pass through the lens. • If there is too much reflected light in the imaging system, this can cause lack of contrast in the image and aberrations. Light reflecting off the sample or other lenses can also create glare. By using anti-reflective coatings, glare is reduced and contrast in the image is improved.

5.6 Prisms Prisms are used extensively in microscopy to redirect or split light. There are different types of prisms, and they are classified based on their shape and function. Dispersive prisms are used to separate the colours of light. The reflective prisms—as the name would suggest—reflect the light and send it in a different direction. Reflective prisms are primarily used for redirecting the light into the microscope eyepieces or CCD (Fig. 5.3). What is the difference between a lens and a prism?

Fig. 5.3  Prisms are geometrically shaped pieces of crystal or glass that are used to redirect light

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• A lens can focus and magnify light, whereas a prism can redirect and split light without changing the focus or magnification of the light rays. • A prism has at least five flat sides. There are no curved sides on a prism, whereas a lens will have curved sides. • To be technically correct, a prism should have two matching sides.

5.7 Magnification Magnification defines how much the sample appears to have increased in size. A 10× objective makes the sample appear ten times larger, a 20× objective makes the sample appear twenty times larger, etc. A good quality magnifying lens should increase the apparent size of the sample, but not distort the shape or proportion of the object being viewed. Magnification is a dimensionless number, which means that a physical measurement cannot be ascribed to it. What is being measured is the angle by which the sample is being subtended. In microscopy, the magnification value is a combination of the objective magnification plus the eyepiece magnification. So, if the objective magnification (Mo) is 10× and the eyepiece magnification (Me) is 10×, the total magnification (MT) is 100×. The equation for total magnification is therefore: MT = Mo x Me. In digital imaging, the magnification of the objective is used to apply the scale bar to the image. Depending on the magnification, the scale bar will be in millimetres (mm) or micrometres (μm).

5.8 Numerical Aperture Numerical aperture (NA) is the ability of a lens to gather light. More specifically, it is the range of angles that light can enter the lens from. All lenses have a NA value, and for microscopy it is the NA values of the condenser and objectives that are the most important. See Sect. 8.5 for more information on NA. • A condenser with a high NA creates a wider cone of light for the sample. • A condenser with a low NA creates a narrower cone of light for the sample (Fig. 5.4). • An objective with a high NA receives a wider cone of light from the sample.

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Fig. 5.4  A condenser with a higher NA creates a larger cone of light (left). A condenser with a lower NA creates a smaller cone of light (right)

Fig. 5.5  An objective with a higher NA receives a larger cone of light from the sample (left). An objective with a lower NA receives a smaller cone of light (right)

• An objective with a low NA receives a smaller cone of light from the sample (Fig. 5.5). • A wide cone of light means a lower light level, as the same number of photons are being spread over a larger area. This is suitable for low magnification viewing. • A narrow cone of light means the photons are more concentrated and the light will appear brighter. This is required for high magnification viewing. • As magnification increases, NA should decrease to make a tighter cone of light and provide the stronger light levels needed.

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5.9 Resolution Resolution in microscopy is how far apart two objects need to be in order for the user to be able to tell that they are indeed, two separate objects. In application, good resolution is being able to tell the number of individual cells in a cluster, or to be able to see the boundary lines between neighbouring cell walls. The strength of a microscopes resolution is known as the resolving power. The higher the resolving power of a microscope, the better its ability to separate out small objects and see fine detail. Resolution is measured in micrometres (μm), and the higher the resolution value, the higher the resolving power. There is a direct correlation between the wavelength of light being used to image and the resolution that can be achieved. As the wavelength of the light decreases, resolution quality increases. The numerical aperture of the objective and the condenser are instrumental in determining the resolution of the microscope. The NA of the objective and the condenser combine to give the total NA of the imaging system. As total numerical aperture increases, so does resolution. Magnification does not correlate with resolution. Resolution is calculated as: R = λ/(2NA), where R = resolution  λ = the wavelength of light, and aperture of the system.

NA = the numerical

In digital images (note, images not imaging), resolution is defined as the number of pixels per square inch. See  Sect.  14.6.4 for more on digital resolution.

5.10 Focus Humans have an innate understanding of focus—when looking at an object, we move it closer or further away from our eyes to see it better. What we are unknowingly doing is adjusting the focal length until the object is at the best focal point for our eyes. Squinting is another behaviour we naturally use to help us focus. By squinting, we are blocking out the lesser focused light rays coming into our eyes from the side, and only allowing the sharper, central light through. In microscopy, we move the lenses up and down to focus the image. By doing this we are adjusting the distances between the lenses until the focal point lands at the correct point for our eyes.

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An image is considered ‘in focus’ when the object being viewed has a sharp and well-defined edge. The entire image should look sharp and all edges should be well defined. This is a primary goal in microscopy—to produce an entirely in-focus image. This is especially important if the images are going to be used for any kind of analysis. Without good focus, correct data cannot be taken from the image. Images that are going to be used for presentations or publications must also be in sharp focus. Firstly, for quality and secondly, if the image is enlarged it will be very obvious if the image is not in focus.

5.11 Aberrations As discussed, the lenses in the microscope refract the light to magnify the image. During refraction, the light can be split into its individual colours. In a microscope, the lenses are aligned in such a way that the light beams can be refracted to magnify the image, but that they will end up together at a perfectly focused point. If the lenses have not been aligned correctly, the light rays will not align properly, and aberrations are created. There are two forms of aberration caused by misalignment of the lenses: –– Chromatic aberrations –– Spherical aberrations • Chromatic aberrations are when  the lenses split the white light and causes the individual colours of light to be seen. • Spherical aberrations are when the lenses are misaligned in such a way that the image cannot focus. • Aberrations are resolved by aligning the lenses correctly.

References 1. Hogg J  (1861) The microscope: it’s history, construction and application. Routledge, Warne & Routledge, London 2. Pfaender HG (2012) Schotts guide to glass. Springer Sci Bus Media ISBN 9401105170, 9789401105170

6 The Anatomy of the Microscope

The simplest pocket microscope...will reveal wonders a thousand times more thrilling than anything which Alice saw behind the looking-glass David Fairchild

Abstract  Most people can very easily recognise a traditional upright microscope. They can also quite easily tell you where to put the sample, how to focus it, and where to look. Now ask them to do the same on an inverted microscope. Or ask them why that upright microscope has a round stage? Microscopes are quite diverse, and different samples require different microscopes. The general anatomy and parts are the same, though. In this chapter, we shall look at the configuration of the traditional upright microscope, the inverted microscope, the stereomicroscope, and the petrological microscope. Each part of the microscope will then be explained over the next several chapters. The key message in this chapter is to note that the different microscopes are just slight variations of each other. Even high-end systems will have the same overall anatomy.

The microscope is one of the most instantly recognisable machines in the world. The overall shape and layout has not changed much throughout the microscopes history and, even today, the configuration of different types of microscopes are built around the same overall layout. Armed with an introductory knowledge of the different parts, and the overall configuration of an © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_6

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imaging system, the user will be able to interpret and navigate most styles of microscope.

6.1 Styles of Microscope There are three general styles of light microscope—the upright microscope, the inverted microscope, and the stereomicroscope. Different types of imaging systems have similar requirements and will be composed of mostly the same parts. The basic configuration of a microscope is described below, and this will describe most models of light microscope. If the user has a good knowledge of the basic configurations described in this chapter, they will be able to approach most microscopes with a certain level of confidence. Note: The best advice when interpreting any new imaging system is to follow the light path. The user can orientate themselves by locating the following: 1. The stage—find where the sample sits. This will usually be in the centre or lower region of the system. 2. The light source—this could be internal or external. See Chap. 11. 3. The eyepiece/camera—find where the image is going to be detected. This is usually at the opposite end of the system to where the light source is. Once these three components have been located, the user should be able to locate all the other parts. For example, the condenser will be between the light source and the stage, the objective will be in close alignment with the sample, etc. The position of the parts of an upright and inverted microscope is outlined below. Each individual part will then be discussed in detail in a separate chapter.

6.2 The Upright Microscope The upright microscope is the traditional configuration and the one that most people associate with microscopy (Fig. 6.1). A note about the model of upright microscope being used here. This microscope is a 1950’s Watson and I chose this microscope to be the model for this book as it was the first antique microscope I bought. It is a classic design and the original light source on it was a mirror. The mirror has been omitted from this image and replaced with a

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Fig. 6.1  Parts of an upright microscope

built-in light source. There are many styles of base and light, so the one here is illustrative. Also note that the focus wheels are at the top, whereas in some models they are lower down. 1 . Eyepieces or camera/CCD—for viewing the image (see Chap. 7). 2. Objective head—rotates so that the user can select the different objective lenses. 3. Objective lens—the primary magnifying lenses (see Chap. 8). 4. Stage—used for holding and navigating the sample (see Chap. 9). 5. Condenser—used for controlling the light as it falls on the sample (see Chap. 10). 6. Diaphragm—used for controlling the amount of light entering the condenser (see Chap. 10). 7. Light source—used for illuminating the sample (see Chap. 11) with transmitted light.

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Advantages of Using an Upright Microscope • The traditional and most common style of microscope, they are easily available and affordable. • Most users have an intuitive ability of how to use an upright microscope.

Disadvantages of Using an Upright Microscope • The objectives have very small working distances, especially at higher magnification. Samples must be very small to fit under the objectives. • The small working distances carry the risk of the slide or container becoming broken if the objective gets too close. • Sample preparation must be of good quality.

6.3 The Inverted Microscope The inverted microscope is a model commonly used in life science research. It has the same overall configuration as an upright microscope, just inverted (Fig. 6.2). Note: Not all inverted systems will have an environmental chamber.

Fig. 6.2  Inverted microscope

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Advantages of Using an Inverted Microscope • Live cells can be imaged in the multiwell plate or petri dish that they have been grown in. This reduces stress to the sample. • There is less risk to the slide. If the objectives get too close to the slide, it will simply push the slide up.

Disadvantages of Using an Inverted Microscope • Oil imaging with an inverted microscope requires some experience.

6.4 The Stereomicroscope Stereomicroscopes, also known as dissecting microscopes, are commonly used for tasks such as dissecting  and manufacturing to see  the specimen more clearly. In manufacturing they are used for tasks such as watch and jewellery making, for seeing better when soldering circuit boards, and for inspecting bonds or parts (Fig. 6.3).

Fig. 6.3  Stereomicroscope

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The magnification is low to medium, usually ranging between 5× and 45×. Reflected light is used to illuminate the sample. Some models will have built in lighting, and others will have external lights, often on goosenecks that can be bent at different angles. Many models have a disc on the stage that can be flipped to be black or white. This allows the user to choose the best background for the sample. High working distance allows the user space to work and use tools. Some models can have a WD of up to 25 cm, depending on the magnification. The advantage of the stereomicroscope is that the high working distance allows the user to handle and manipulate the sample and to do tasks under the microscope. The depth perception achieved in these scopes allows the user to work with correct perspective, which makes accurate hand-eye coordination easier. Stereoscopic viewing is based on the same theory as stereoscopic vision in nature. By having two eyes that are a small distance apart, animals (humans included) see the world from two slightly different angles. The brain then combines these images and creates depth perception. The stereoscopic image created by the stereomicroscope combines two images of the sample from slightly different angles. This gives the image depth, and the specimen under the lens appears three dimensional.

6.4.1 Types of Stereomicroscope There are two varieties of stereomicroscope and they are classified by the design of the objective they have. • A CMO (common main objective) stereomicroscope has one large lens that can be adjusted to give the different magnification. CMOs are better at gathering light, and the image resolution and quality is generally better. • Greenough stereomicroscope uses two lenses aligned beside each other. This gives the user high quality stereo vision of the sample.

6.5 Petrographic Microscope A petrographic microscope is used in geology to investigate birefringence in rock and mineral samples. Petrographic microscopes are easy to spot as they look like regular upright microscope, but they have a round stage that can rotate. Polarising filters are required for petrography (Fig. 6.4).

6  The Anatomy of the Microscope 

Fig. 6.4  Petrographic microscope

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7 The Eyepiece

The task of the right eye is to peer into the telescope, while the left eye peers into the microscope Leonora Carrington

Abstract  In this chapter, we will cover how to correctly adjust the eyepieces, including how to use an adjustable eyepiece, in order to get the clearest field of view. Many users, in their excitement to see their sample, neglect the eyepiece. One of the most common complaints I have heard from beginners is that they find eyepieces uncomfortable to use. This is usually because they are not positioned correctly. In this chapter, we will discuss how to have correct interpupillary distance, how to adjust the height of the eyepieces for comfortable viewing, and how to make using eyepieces more comfortable for glasses wearers.

The function of the eyepiece (also known as the ocular) is to focus the light being sent from the objective for viewing by the eye of the user. The structure of an eyepiece can be considered to be a simpler version of the objective. There can be several lenses inside an eyepiece, but simpler models may only have one or two. The lens that is closest the users eye is called the ‘eye lens’, and the lens nearest to the objective is the ‘field lens’. Early models of microscope such as the Leeuwenhoek microscope did not have eyepieces, and the user viewed the sample directly through the main magnification lens. The first compound microscope designed by the Jansens in the sixteenth century had an eyepiece © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_7

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lens for extra magnification. For much of the microscopes history, there was only one eyepiece (monocular). Models such as the Culpeper microscope, and the tube microscopes of the seventeenth and eighteenth century, had a single eyepiece with a magnifying lens for the user to comfortably observe the sample. In the nineteenth century, the technology of microscope making excelled and binocular eyepieces became a standard design. The first binocular microscopes used a prism to split the image between the two eyepieces, and this method is still used today. In the older models,  the prism would often be removed for viewing samples at higher magnification, directing the full resolving power to one eyepiece [1], however modern models have overcome this through improved optics. Eyepieces not only deliver the image to the user, but they also add extra magnification to the image and the most common eyepiece magnification is ×10. Therefore, the total magnification of an image should be considered to be the objective magnification combined with the eyepiece magnification. For example, if the user was viewing a sample using a ×20 objective and ×10 eyepieces, the user is viewing the sample at ×200. When using a ×40 objective and ×10 eyepieces, the user is viewing the sample at ×400. Some digital systems that use a camera or CCD have been designed to add ×10 magnification to the image to replicate the appearance of the sample as if it were being viewed through eyepieces.

7.1 Compensating Eyepieces The eyepieces on a microscope should be considered to be an integral part of image formation. The construction of an eyepiece—while simpler than an objective—is just as considered. Eyepiece lenses can be treated for correcting the appearance of chromatic and optical errors that might be created during magnification. Some models of eyepiece are designed to work in conjunction with particular models of objective, and these are known as compensating eyepieces [2].

7.2 Eyepoint For the image to be seen correctly, the users eyes must be at the correct height from the eyepiece lens (i.e. the focal point of the light coming from the sample should be landing on the retina). The position that the users eyes must be in above the eyepieces is known as the eyepoint or eye relief. Eyepieces can be high-eyepoint or low-eyepoint.

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A common mistake made by first-time users is that they position their eyes too close or too far from the eyepieces. If the eyes are too far away, the user will see the image as two circles instead of one circular, binocular image. If the eyes are to close, the user will have a wonderful view of their eyelashes. The correct eyepoint will be recognised as the point at which the user sees one perfect circular field of view (sans eyelashes).

7.3 Interpupillary Distance The interpupillary distance is the distance at which the users pupils are perfectly aligned with the centre of each eyepiece lens. Interpupillary distance can be easily adjusted on most models of binocular microscope by simply moving the eyepieces further apart or closer together as required. It is a common mistake in first-time users to not correctly adjust the interpupillary distance. It can be recognised as seeing two circular field of views instead of one perfect circle. Every user is different, so checking the eyepiece settings at the beginning of an imaging session should be part of the users good practice (Fig. 7.1).

7.4 Using Eyepieces as a Glasses Wearer Microscope eyepieces can be classified as high eyepoint or low eyepoint. A user that wears glasses will find that their eyes are further from the eyepiece than a user who does not wear glasses. This will result in trouble viewing the

Interpupillary distance

Fig. 7.1  Having the correct interpupillary distance is vital for clear vision

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image properly and could result in focus issues. Therefore, high eyepoint eyepieces are designed for users who wear glasses, and low eyepoint eyepieces are for users who don’t wear glasses. Rubber cups on the eyepieces can be folded up or folded down in accordance with the distance the user sits from the microscope. Again, every user is different and should adjust the eyepieces as much as needed to get a clear image.

7.5 Diopter Eyepieces Many people have slightly different visual acuity in each eye. This can result in the focus being different in two equally set eyepieces. To allow for natural variation between the eyes, a dioptre eyepiece can be used. The normal configuration is that there will be one fixed eyepiece and one adjustable eyepiece (Fig. 7.2).

7.5.1 How to Adjust a Diopter Eyepiece 1. With a sample on the stage and the light source on, first ensure that the interpupillary distance is correct. See above for how to adjust the interpupillary distance.

Non-adjustable eyepiece

Adjustable eyepiece

Fig. 7.2  Microscopes with binocular eyepieces often have one eyepiece that is adjustable and one that is not. The adjustable eyepiece is known as a diopter eyepiece. This is to allow for variation in the visual ability of each eye

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2. Close the eye that is looking down the diopter eyepiece. While only looking down the fixed eyepiece, use the focus knobs to bring the sample into focus. 3. Once the fixed eyepiece image is in focus, close that eye and open the eye on the diopter eyepiece side. 4. Adjust the dial on the diopter eyepiece (not the focus knobs)  until the image comes into focus. 5. Open both eyes and look down the eyepieces as normal. The image should now be in focus for both eyes equally. The eyepiece focus might need to be adjusted over different magnifications, but adjustments will usually be minor.

7.6 Reticle A reticle (also known as a graticule) is a glass disc insert that has a scale bar, grid, or micrometer etched into it. The reticle is placed in the eyepiece and provides the user with a means of measuring or counting features in the sample. As the reticle is situated in the eyepiece, it does not change when the objective (and therefore the magnification) is changed. A sample viewed at ×10 might measure 0.5 mm, and the same sample at ×20 might measure 1 mm. This is when adjusting for scale and being able to calibrate the reticle becomes essential knowledge for the microscope user (Fig. 7.3).

Fig. 7.3  There are a variety of different reticles available. Some aid the user in counting cells, some are for measuring and are available in a range of different scales

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7.6.1 How to Calibrate a Reticle Reticles are calibrated using a glass slide called a micrometer. A micrometer is a glass slide that has a highly accurate scale bar etched or printed onto it. Precision micrometers can be extremely expensive, so they must always be handled with care. Note: Pay careful attention to what the scale of the micrometer is. Note the total measurement and the measurement of each division. For example, if 100 divisions is equal to 1 mm, therefore each division is 10 μm. This is essential information for accurate calibration. 1. Place the micrometer slide on the stage. Start at the lowest magnification on the microscope and bring the scale on the micrometer into focus. 2. Looking down the eyepieces, align the micrometer on the stage with the scale bar/grid on the reticle in the eyepiece. 3. Count the number of reticle divisions that align with a set value on the micrometer. For example, how many reticle divisions equal 100 μm on the micrometer. 4. Divide the number of reticle divisions by  the number of μm on the micrometer to calculate what each reticle division measures. 100 divisions = 1 mm 1 division = 10 μm

Micrometer scale (on the stage)

Reticle (in the eyepiece)

20 reticle divisions = 100 μm divisions 100 μm / 20 = 5 μm Therefore one reticle division = 5 μm

7.7 Cleaning an Eyepiece The procedure for cleaning the eyepieces is quite simple. As part of good practice, the eyepieces should never be removed from the microscope, as this could let dust and dirt into the system. The most common requirement will be cleaning the rubber eyecups. The eyecups are one of the only parts on the microscope that comes into regular contact with the user and therefore they can get quite dirty. Oils from the skin, sweat, and makeup can be easily cleaned from the eyecups using an alcohol wipe or a glasses wipe.

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A glasses wipe is best for cleaning the eyepiece lenses. The user must be conscious not to use too wet a wipe, as if fluid gets inside the eyepieces they will be ruined. Always wring out a wipe before applying it to the lenses—if it drips when you wring it, it is too wet. See Sect. 15.9 for the Wring Test and see Sect. 8.15 for how to clean a lens.

7.8 Scale Bars on Digital Systems In digital systems the software can be told which objective is in which position on the objective head. This way the software knows what magnification is being used, and it can apply a scale bar accordingly. However, the user must still use their judgement. Objectives can be swapped around, software can be calibrated incorrectly. The user needs to be able to judge if the scale bar is wrong. If it is, then the software needs to be calibrated. Refer to your systems user guide on how to calibrate the software.

References 1. Turner G (1981) Collecting antique microscopes. Christies South Kensington Collectors Series. ISBN 0289708826 2. Akinyemi O, Boyde A, Browne M, Hadravsky M, Petran M (1992) Chromatism and confacality in confocal microscopes. Scanning 14:136–143

8 The Objective

People think of science like somehow that’s the answer, and that it’s all about right answers, but science is a lens that we look at the world through Dallas Campbell

Abstract  The objective is one of the most expensive and influential parts of your microscope. So, a knowledge of how to use your objective optimally is an essential weapon in the microscoper’s armoury. In this chapter, we will go through the different codes that you will see on an objective and what they mean. This will enable you to choose the right objective for the job and help you get the best from your equipment. We will also cover how to use and clean air and oil objectives.

The objective is the eye of the microscope. It captures the light from the sample, magnifies and focuses that light, and then sends it on to the eyepieces for the user to see. The objective is one of the main players for magnification, in addition to the eyepieces. See Chap. 7 for more on eyepieces. Most microscopes will have several different objectives, each with a different magnifying power. The objective is usually one of the most expensive parts of the microscope, so it is important to take good care of it (Fig. 8.1). An objective is a precision piece of engineering. It is composed of several lenses in a casing, manufactured in a clean room to ensure that no dirt, dust, or moisture is inside. Everything about these lenses will have been calculated by an optical engineer to ensure that they work in perfect synchronicity— © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_8

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Fig. 8.1  A microscope will have a range of objectives of a variety of magnifications

from the curvature of the lenses, the distance between them, and any coatings being used. The two most common types of objective for light microscopy are air objectives and oil objectives. Knowing the difference is important for the correct use and care, and we will discuss both of these here. In this chapter the specifications of the objectives and how to read the coding on the objective will be covered, as well as how to use and maintain the equipment. This section should be used in conjunction with Chap. 5. This will help the user understand how the objective works but also to help the user troubleshoot any imaging issues that may be met. A few important notes before starting. • Store any objectives that are not being used in the packaging they came in. Keep them somewhere cool and dry—humidity can cause a lot of damage to an objective.

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• When handling the objective, always hold it by the sides of the casing and avoid touching the glass. • Try to avoid removing the eyepieces or objectives from the microscope. When parts are removed from the microscope, it increases the chance of dirt and dust getting inside the tubing. Dust inside the tubing can be difficult to get rid of and will affect image quality. • Never try to take apart an objective! If fluid or dirt of any kind gets inside an objective—or if any of the lenses are skewed—the objective is ruined. If an objective needs to be repaired, this must be done by a professional.

8.1 How to Read an Objective There are lots of great resources available to teach the user how to read and understand the codes on microscope objectives. The reason for this is quite simple—it’s important! A good rule to follow is the more information that is written on the objective, the better quality (and more expensive) the objective is. Understanding this information will help the user choose, use, and get the best results out of the objective.

8.1.1 Achromatic and Apochromatic When white light passes through a lens, the different colours of light are refracted to different extents (see  Chap. 4, for more on this). To create a good quality image, all the different colours of light must converge on the same focal point. If the different colours of light do not manage this, an effect called a chromatic aberration can be seen (see Sect. 5.11 for more on chromatic aberrations). This occurs because the different colours of light have been dispersed in such a way that they are forming their own individual focus points. This is usually caused by misalignment or incorrect shape of the lenses. A chromatic aberration can be recognised as the image being (a) slightly out of focus and (b) colours (usually red and blue) appearing around the edge of the sample or field of view. Achromatic and apochromatic lenses correct for this behaviour. • Achromatic and apochromatic lenses are lenses that have been designed to make sure that all the colours of light stay together and all converge on the same focus point.

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• This will be noted on the objective using the abbreviation ‘achro’ for achromatic and ‘apo’ for apochromatic. • Achromatic (achro) lenses correct for red and blue misalignment. • Apochromatic (apo) lenses correct for red, blue, and green misalignment.

8.1.2 Infinity Correction For an image to be in focus, the light rays have to come together at a particular point, known as the focal point. For the user to see a focused image, that focal point needs to be at eye level or at the level of the CCD (see Sect. 14.6.2 for more on CCDs). If any extra parts (such as a camera) are added to the optical path, the distance between the objective focal point and the focal point for the user is increased. Infinity correction means that extra attachments (such as a camera) can be added to the light path and not interrupt the focus. How does infinity correction work? An extra lens is added to the light path that extends the light rays and allows for the image to focus at the correct point for the user. Infinity correction is indicated on the objective by the infinity symbol ∞

8.1.3 Magnification For a full explanation of magnification, please see Sect. 5.7. Magnification is noted on the objective as a number and the letter ‘×’. For example, a 10x  (or x10) denotes that the objective will magnify the sample by 10.

8.1.4 Numerical Aperture (NA) For a full explanation of numerical aperture, please see Sect. 5.8.  Numerical aperture is noted on the objective as NA and a value. For the best resolution, the NA value of the objective must match the NA value of the condenser.

8.1.5 Planar In order to bend light, the lenses in an objective are curved. While this curvature is the feature that allows the lens to magnify and focus images, it also

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causes an effect called vignetting. Light rays passing through the centre of the lens are not refracted very strongly. The light rays passing through the edges of the lens—where there are  two, more acutely angled surfaces to pass through—are refracted quite strongly. All lenses will experience some level of vignetting, and most microscopes are designed in such a way that the areas of vignetting are not in the field of view. • Vignetting is recognised as the centre of the image being well lit and in focus, while the edges of the image are darker and out of focus. • Vignetting is an example of a spherical aberration. • A planar lens has more flat area in the centre of the lens. This means that any vignetting gets pushed further outwards and out of the field of view. Planar lenses are indicated by the code ‘plan’ on the objective. The higher the ‘plan %’, the larger the flattened area and the less vignetting that the user will see (Fig.  8.2).  In many imaging systems, the FOV of the eyepiece is slightly less than that of the objective. By concealing the very edges of the objective FOV, any vignetting that is around the very edges of the image is hidden from the user. So, while some vignetting is occurring, it does not affect the image.

Fig. 8.2  Vignetting can be Vignetting can be seen as the reddish-brown line around the edge of the field of view. This area will be out of focus and should be excluded from the image where possible

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8.1.6 Refractive Index (RI) For a full explanation of refractive index, please see Sect. 4.5.  When imaging with an oil immersion objective, the user needs to apply immersion oil. The refractive index of the oil must match the refractive index of the objective. Refractive index will be noted on the objective as an RI and a value. The RI value of glass is approximately 1.3; the RI value for oil can be up to approximately 1.5. When buying immersion oil for imaging, it is not the brand that is important—it is the RI value (the RI of the oil should match the RI of the oil objective).

8.1.7 Thread Depth and Brand Compatibility Objectives are threaded for a secure and stable attachment to the objective head. The Royal Microscopical Society (RMS) defines the standard thread depth (or thread pitch) as 0.706mm. This was established in 1858, making measuring the thread depth a way of dating objectives on older machines [1]. If the objective matches this standard, it will be marked as ‘RMS’. Any objective with standard threading can be used on any standardised microscope. Certain brands, however, use a slightly different thread  depth. This means that objectives of other brands will not fit on their microscopes and that only their parts can be used on their machines. • The diameter of the threading on the objective is listed as an ‘M’ number, as it is measured in metric. For example, M32 is an objective with a 32 mm diameter. When purchasing a new objective, the thread depth will be listed in the technical specifications.

8.1.8 Working Distance (WD) The working distance is defined as how close the objective lens needs to be to the sample in order to focus. When working at higher magnifications (×60, ×100), the objective will get very close to the slide. If the user is not careful while adjusting the focus at high magnifications, the objective can crash into the slide and break it. • The higher the magnification, the lower the WD.

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• High magnification objectives are usually sprung—this means that they will depress when pressure is put on the front of the lens. If the objective contacts the slide, the objective will flex instead of crashing through the slide. • The working distance of the objective will be indicated by the letters WD and is measured in millimetres. Below is a table listing the approximate WD values for the most common objective magnifications. These are approximate values, so the user should always refer to the actual WD value on the objective that they are using. Objective magnification

Working distance (WD)

×5 ×10 ×20 ×40 ×60 ×100

34 mm 4.0 mm 0.35 mm 0.20 mm 0.21 mm 0.13 mm

8.2

Air Objective

An air objective is the standard type of objective  and the most commonly used. The light travels from the sample and through the air before it meets the objective lens. As light moving through air is subject to scattering, air objectives achieve low to medium levels of magnification. • The light travels through air from the sample and into the objective lens (Fig. 8.3). • This is the most common type of objective for light microscopy. • Magnification range is low to medium, due to light being  scattered by the air. • The most common magnifications for air objectives are ×5, ×10, ×20, and ×40.

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Fig. 8.3  Light travels through the sample, through air, and into the objective

8.3

Oil Immersion Objective

An oil immersion objective is used for higher magnification imaging. By adding immersion oil, we prevent the light from scattering, essentially using the oil to funnel the light into the objective lens. • The light travels through immersion oil from the sample and into the objective lens. • Magnification range is medium to high. • The most common magnifications for oil imaging are ×60 and ×100. • Oil objectives are usually sprung (see Sect. 8.9). • Requires immersion oil to be added to the slide for imaging. • RI value of the immersion oil must match the RI of the oil objective (see Sect. 8.7). • Water immersion objectives are also available. The RI of the lens matches that of water instead of oil. Treat them the same as an oil objective (Fig. 8.4).

8.3.1 How to Use an Air Objective Air imaging is the most common type of imaging in microscopy. It  is the technique that most users learn first and also a technique that is  used

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Fig.  8.4  Light travels through the sample and is directed by the oil  into the objective

in most areas of research. By the time the user is graduating onto water or oil imaging, they should be well experienced in air imaging. 1. Position the microscope on a sturdy and steady surface. Switch on the light source (some light sources will need a few minutes to warm up. See Chap. 11 for more on light sources). 2. The user should be seated comfortably and ensure that the eyepieces are at the correct eye level. Ergonomics is important when using a microscope. If the eyepieces are too low the user will hunch, and this will result in back and neck pain. If they are too high, the user will not be able to see the image properly. 3. It is good practice to start at the lowest magnification objective and work up to the higher ones. This allows the user to skim over the sample first and review it before moving to the higher magnifications, where the FOV will be reduced. 4. Place the prepared slide on the stage and secure it using the slide clips. The coverslip of the slide should always be facing the objective lens.  This is important to remember if using an inverted system—using an inverted system - where the slide is placed with the coverslip facing down. 5. Watching the objective (i.e. not looking down the eyepiece) use the coarse focus to bring the sample as close to the objective as possible, without them actually touching. By starting in this position, the chances of breaking the slide with the objective are reduced, as the user will be moving the

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sample away from the objective to focus, rather than towards it. This is especially important at higher magnifications, where working distances are minute. 6. Look through the eyepieces and start to slowly move the sample away from  the objective using the coarse focus wheels until the image comes into focus. The fine focus wheels can then  be used to make small adjustments. When using the focus wheels, the user is moving the sample in the Z-axis. 7. Once the image is in focus, the stage controls are used to navigate around the sample. If the sample is well-prepared and the stage is secure (i.e. moving in the X axis and Y axis without drifting in the Z axis), the user should not need to adjust the focus too much during imaging. 8. When moving between objectives,  ensure that there is sufficient space between the stage and the objective for the objectives to rotate. Select the next objective by turning the objective head. Most objectives will click when they are in the correct position. Return to step 5 and repeat the steps again. Remember that looking after the sample is just as important as looking after the microscope!

8.3.2 How to Use an Oil Immersion Objective Using an oil (or water) immersion objective is the same as using an air objective, but there are a few small and important differences. 1. Position the microscope on a sturdy and steady surface. This is especially important for oil imaging as (a) fluid is being introduced to the system and (b) the user is working at high magnification. At high magnification, even small vibrations can have a big impact on image quality. Switch on the light source (some light sources will need a few minutes to warm up. See Chap. 11 for more on light sources). 2. The user should be seated comfortably and ensure that the eyepieces are at the correct eye level. Ergonomics is important when using a microscope. If the eyepieces are too low the user will hunch, and this will result in back and neck pain. If they are too high, the user will not be able to see the image properly. 3. It is good practice to start at the lowest magnification objective and work up to the higher ones. This allows the user to skim over the sample and review

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it before moving to the higher magnifications, where the FOV will be reduced. For oil imaging, the lower magnification is usually around ×60 and then moves up to ×100. The sample will usually have been examined with lower magnification air objectives first, to ensure sample quality. Bad sample prep is even more obvious at high magnification (see Chap. 13 for more on sample Prep). Important! If the microscope has a combination of air and oil objectives, pay very careful attention to what type of objective is being selected. Oil objectives will not work without oil, and if immersion oil is used with an air objective it will destroy the air objective. If moving from an oil objective to an air objective, clean the sample slide and oil objective first (see Sect. 8.14 for more on cleaning an objective). 4. There are two techniques for placing the slide and adding the oil. Both have pros and cons, so the user should use the technique they feel most confident with. The important thing to remember is that the oil needs to be applied to the slide without getting it anywhere else. Oil in the condenser means buying a new condenser!

(a) The first technique is to apply the drop of oil to the coverslip first and then position the slide on the stage. The advantage of this is that there is only a small amount of fluid that can possibly spill. The disadvantage is that the slide must be kept perfectly level and steady while placing it on the stage and securing the slide clips. This is the technique that has to be used for inverted imaging. (b) The second technique is to place the slide on the stage and secure it under the clips first and then apply the oil. The advantage of this is that the slide can be secured in place without worrying about displacing the oil. The disadvantage is that if too much oil is applied to the slide, it will need to be removed from the stage and cleaned. The other disadvantage of this is that oil could be accidentally spilled down into the condenser and light source during application, which will destroy them. 5. Important! This step is very delicate. Do not rush it! Centre the oil drop over the light path using the stage navigation controls. Watching the objective (i.e. not looking down the eyepiece), use the coarse focus dials to bring the objective just above the oil drop (Fig. 8.5a). Very slowly and carefully, use the fine focus dials to lower the oil objective until it comes into contact with the oil drop.

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Fig. 8.5  The oil drop must be approached slowly. Once the objective has contacted the oil, then imaging can begin

There are two ways to recognise this moment—(a) the oil drop will catch onto the objective  and it will go from dome-shaped to a column shape, and (b) the oil will light up (Fig. 8.5b). Once the objective has contacted the oil, only use the fine focus wheels. Look down the eyepieces and adjust the focus in tiny increments. 6. Once in focus, use the stage controls to move around the sample. If the sample is well-prepared and the stage is secure (i.e. moving in the X axis and Y axis without drifting in the Z axis), the user should not need to adjust the focus too much during imaging. Oil immersion imaging will cover smaller areas than air imaging—do not rush. 7. The heat from the light will thin the oil over time. As the user is exploring the sample, the oil will spread across the coverslip area. Over time, this loss of viscosity and spreading of the oil will cause the oil to lose its light channelling ability—this will cause the image to lose focus. Remove the slide (carefully), clean the objective as per the instructions in Sect. 8.14.  Clean the slide  with an alcohol wipe and dry it. Reapply fresh oil, following the chosen technique from step 4 and repeat step 5. 8. When moving between objectives, only move between oil objectives. Bring the current objective to its highest point (i.e. furthest away from the sample) and clean it as in step 7. Select the next oil objective to be used. Go back to step 4 and repeat the steps again. Remember, extra care must be taken during oil imaging, of both the microscope and the sample. 9. The oil immersion objective should be cleaned after every use. Use the instructions in Sect. 8.14 to clean the objective correctly.

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Cleaning an Objective

Maintenance on light microscopes is fairly minimal and mostly requires just a small tidying up after use. Microscopes in working environments will usually have an annual service and this is often all that is required to maintain them. It is usually the eyepieces—or rather the eyecups on the eyepieces— that need the most regular cleaning, as they are the only part that actually comes into extended contact with the user (other than the focus knobs).  Air objectives should rarely need to be cleaned. If used correctly, the lens of an air objective will never come into contact with anything. The most common reason to clean an air objective is to remove dust. An oil objective, however, should be cleaned every time it is used. If not cleaned after each use, the oil will build up on the lens. Dust gets stuck to the oil and, over time, this will build up and harden on the expensive oil immersion objective. The inside lens of an objective never needs to be cleaned.

Important! • Eyepieces and air objectives are not sealed—this means that fluid can get inside them. Never use anything wet to clean them!. Oil immersion objectives are sealed around the outside lens, but caution is still required when cleaning. If moisture gets inside an objective, it is ruined! • Remember, if using an alcohol wipe, always do the wring test first! (Sect 15.9). If no fluid drips when the wipe is tightly wrung, then it is safe to apply to the objective. • When wiping any of the glass on the microscope, always wipe in one direction, do not use a scrubbing motion. If there is debris on the lens, a scrubbing motion will take that debris and scratch it back and forth across the glass. Coatings on lenses are incredibly thin and can be easily scratched (see more about coatings in Sect. 5.4). • If it is absolutely necessary to take an objective off the microscope for cleaning—for example, if it can’t be reached while on the head—hold the objective by the casing and clean it while holding it pointing downwards (i.e. the outer lens pointing to the floor and clean it from below). This way, any fluid or dirt will fall towards gravity instead of seeping into the objective.

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8.4.1 Cleaning an Air Objective Equipment –– A glasses cloth or dry lens tissue –– An alcohol wipe or glasses wipe if the lens is particularly dirty (e.g. if there are fingerprints on the glass).

Method 1. Switch off any electrics and ensure the bulb has cooled before doing any maintenance on the microscope. Remove any slides or samples from the stage. 2. Leave the objective on the microscope. 3. Use the coarse focus to move the stage away from the objective—this is to give more room to work. 4. Use  the cloth or wipe to clean the outside lens of the objective, wiping gently in one direction. Do not use a scrubbing motion.

8.4.2 Cleaning an Oil Objective Equipment –– A piece of dry lens tissue –– An alcohol wipe or glasses wipe Note: Immersion oil becomes thinner as the light source warms it. Try and clean an oil objective as soon as possible after use, while the oil is still warm and thin. Oil that has dried onto an objective is harder to remove. If cleaning dried oil off an oil objective, be patient. Slowly work the oil off with an alcohol wipe as described in step 4.

Method 1. Switch off any electrics and ensure the bulb has cooled before doing any maintenance on the microscope. Remove any slides or samples from the stage, being careful not to spill any oil. 2. Leave the objective on the microscope.

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3. Use the coarse focus to move the stage away from the objective—this is to give more room to work. 4. Using a piece of dry lens tissue, wipe the outside lens of the objective, wiping gently in one direction. The oil will stain the tissue. Repeat, using a clean part of the lens tissue each time, until no more oil stains are seen on the tissue. Once the lens tissue is coming away clean and dry, wipe the lens with an alcohol wipe.

8.5

Summary of Objective Codes

Objective codes Achromatic Red/blue corrected to prevent aberrations Apochromatic Red/blue/green corrected to prevent aberrations Infinity correction Allows extra parts to be added to the light path without losing focus Magnification How much the object is perceived to have been enlarged by Numerical The range of angles that the lens can accept light  aperture Planar The % of flattened area in the centre of the lens, reduces vignetting Refractive index The extent to which the lens refracts light Thread The thread of the objective must match that of the microscope Working distance The distance the objective lens must be from the sample in order to focus

Achro Apo ∞ X  NA Plan% RI M WD

Reference 1. Turner G (1981) Collecting antique microscopes. Christies South Kensington Collectors Series. ISBN 0289708826

9 The Stage

Science is a collaborative enterprise, spanning the generations. When it permits us to see the far side of some new horizon, we remember those who prepared the way—seeing for them also Carl Sagan

Abstract  The stage is the unsung hero in microscopy. Without a perfectly level and controlled stage, imaging is a chore and keeping the sample in focus is incredibly laborious. In this chapter, we will look at how to focus the image and how to navigate around the slide. We will also compare the stages in an upright microscope, with those of a petrological microscope, and an inverted microscope. The stage is where the sample will sit during imaging. It is often an overlooked part of the microscope, but it is more than just a platform. A properly functioning stage is essential for good focus and for comfortable and effective navigation of the sample. Important characteristics of the microscope stage are: • The stage must be perfectly level in the X and Y axis. If the stage is not perfectly level, this will cause focusing issues while navigating the sample. • The stage must be able to hold its position in the X, Y, and Z axis (Fig. 9.1) when the user is not holding the stage controls or focus knobs. If the stage cannot hold its position in the Z axis, this will cause issues with focus. • If the stage cannot hold its position, this is called ‘drifting’. © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_9

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Fig. 9.1  The stage must be perfectly level and be able to hold its position in the X, Y and Z axis for smooth viewing of the sample

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9.1 Focusing the Sample Some models of microscope focus by moving the objective to the sample, and some focus by moving the stage to the objective. Moving any part of the microscope vertically up or down is known as moving it in the Z axis. The Z axis movement is used for focusing the image but also for a technique called Z-stacking, which is discussed in Chap. 12. • Focusing the image is done using the focus knobs which are found on the body of the microscope. • There will be two sets of focus knobs—one for course focus and one for fine focus. • The course focus knobs are for making larger movements, such as bringing the objective down to the stage (or vice versa) or for lifting the objectives away to make space. Note: When using the course focus knobs, the user should be watching the stage, not looking down the eyepieces. This will prevent the user from accidentally contacting the slide or stage with the objective. • The fine focus knobs are used for smaller movements, such as the final approach to the slide and small adjustments during imaging. Note: When using the fine focus knobs, the user should be looking down the eyepieces. The fine focus knobs should be turned in small increments to avoid crashing the objective into the slide. The user should familiarise themselves with the microscope by exercising the dials in each direction and observing the movement.

9.2 Slide Navigation Controls Moving the sample on the stage is done using the slide navigation controls. These are located either underneath or beside the stage. • Navigating the sample is mostly done while the user is looking down the eyepieces, so it is a good idea to be familiar with the steering of the stage controls and slide navigation controls. • Many microscopes use counter-steering—i.e. when looking down the eyepiece, steering  the sample to the left appears to move the sample to the right when looking down the eyepieces. • Navigating around the sample will only require very small movements of the stage navigation controls, especially at higher magnifications.

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• Move around the sample slowly, it can be easy to get lost and to lose interesting features in the sample. The user should familiarise themselves with the stage navigation controls by exercising the dials in each direction and observing the movement of the slide holder.

9.3 Petrographic Microscope Stage Geological samples can be viewed on a regular light microscope stage, but scientists who are investigating birefringence require a circular, rotating stage. This type of microscope is called a Petrographic microscope. See Sect. 6.6 for more on Petrographic microscopes. The sample is centred on the rotating stage and focused using the course and fine focus. The sample is then slowly rotated. As the sample rotates, any birefringent areas in the sample will be seen to change colour. Course and fine focus are done using the same method as a regular microscope, and the stage is rotated by hand. For the correct observation of birefringence, the objective must be perfectly centred on the stage. For instructions on how to centre the objective, see Sect. 15.10. Important characteristics of a Petrographic microscope stage are:

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• The stage is round and rotates 360° in both directions. • The sample sits in the centre of the stage. • The stage moves in the Z-axis for focusing, but does not move in the X and Y-axis. • Markings on the edge of the stage denote 1 arc of degree.

9.4 Stages for Inverted Microscopes Inverted microscopes are predominantly used in life science research. Inverted microscopes are used because they allow the sample to be imaged in the container that they have been grown in. See Sect. 6.3 for more on inverted microscopes. Being able to image the cells in the container they grew in allows for more sensitive and natural imaging of the sample. Important characteristics of an inverted microscope stage are:  • Stage controls are used to move the sample in the X and Y axis. • Focus knobs move the objective up to the sample to focus.

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• To accommodate the containers, inverted microscope stages will have a number of different inserts available that will fit a range of different sized petri dishes and multiwell plates, as well as slides. • Specialised stages such as heated stages can be used for maintaining the sample. Environmental chambers for controlling CO2 and humidity levels can also be fitted.

10 The Condenser and Diaphragm

A great discovery is a fact whose appearance in science gives rise to shining ideas, whose light dispels many obscurities and shows us new paths Claude Bernard

Abstract  Essential to the quality of your image, the condenser can often be a source of confusion for the new microscoper. Mastering how to correctly adjust the condenser and diaphragm is not difficult. They allow you to optimise contrast and light levels, which will greatly improve the quality of your image. In this chapter, we will look at the anatomy and functionality of the condenser and diaphragm separately and how to make sure they are correctly positioned.

Controlling the amount of light that is reaching the sample is an important part of obtaining a good image. Too much light (overexposure) or too little light (underexposure) will result in an image where there is poor contrast and incorrect colour representation. The level of light can be adjusted at the light source, but better control can be found from using the diaphragm and condenser. The condenser also plays a vital role in the clarity of the image, and thus, it is an important part to understand and know how to use correctly. The iris diaphragm was invented by Robert Hooke [1], the writer and ­illustrator of Micrographia—one of the first and most famous books on microscopy. © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_10

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Fig. 10.1  The condenser and iris diaphragm control the amount of light reaching the sample

The first condensers were used in the seventeenth century and they consisted of small glass globes to focus the light onto the sample. The condenser as we know it now is believed to have originated in France in 1837 and came into common use around Europe by the 1840s [2]. Correct focus and ­alignment of the condenser  greatly increased the resolution of microscopes and it quickly became the fixture that it is today (Fig. 10.1).

10.1 The Diaphragm The diaphragm is used to control the amount of light that is entering the condenser and ultimately the amount of light that is reaching the sample. In standard configurations, the diaphragm is located between the light source and the condenser. In photography that uses SLR cameras, the signature clicking noise that is heard when a photograph is captured is the sound of the cameras diaphragm opening and closing at speed. In microscopy, the dia-

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Fig. 10.2  Disc diaphragm

phragm stays open during viewing and imaging, but the size of the aperture (the opening) can be adjusted. There are two types of diaphragm—the disc diaphragm and the iris diaphragm.

10.1.1 The Disc Diaphragm The disc diaphragm is a rotating disc or slider with apertures  of different diameter. The larger apertures  allow through more light, allowing for a brighter image. As the apertures  get smaller, the amount of light passing through is reduced. The user rotates the disc (or slides the slider) to align one of the apertures  with the light path. This is a simpler and more affordable model of diaphragm and is mostly seen on antique, student, or toy microscopes. The advantage of this model is the simplicity of it—the user simply chooses the fixed-size aperture that gives the desired light level. The other advantage is the lack of moving parts—if there are no moving parts, there is nothing to break. The disadvantage is that the user can only use fixed levels and there are no in-between levels (Fig. 10.2).

10.1.2 The Iris Diaphragm The iris diaphragm is the more advanced—and now more common—style of diaphragm. It is a ring that contains a set of overlapping leaves and it looks much like the diaphragm that is seen down a camera lens. The leaves slide over each other to open and close the aperture and are controlled by a small lever that the user can adjust manually. When fully open, the leaves disappear into

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the ring and the light will be very bright on the sample. As the iris closes, the level of light is reduced. When fully closed, the light is entirely blocked from the sample. Some models do not close completely and when the lever is fully in the closed position, there will still be a small gap allowing a minimal amount of light through. The advantage of the iris diaphragm model is that the user can achieve finer tuning of the light levels. The user can adjust the iris by tiny increments to get the light level just right. The disadvantage (other than being more expensive) is that the leaves of the diaphragm can become misaligned. If this happens then the diaphragm will not work properly and the light distribution will be uneven across the field of view. If the leaves do become misaligned, they can usually be fixed by gently pulling them back into their correct position. If the leaves are loose or buckled, they will continue to be a problem though (Figs. 10.3 and 10.4).

Leave of the iris diaphragm

Fig. 10.3  The iris diaphragm opens and closes to adjust the amount of light entering the condenser

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Fig. 10.4  The Iris diaphragm is composed of a number of over-lapping leaves that are opened and closed to precisely control the level of light reaching the condenser

10.1.3 How to Centre the Iris Diaphragm It is essential that the diaphragm be correctly centred so that the sample is evenly illuminated across the field of view. This is an easy setting to check and should be part of good practise when starting an imaging session. Note that some diaphragms are not adjustable. If the diaphragm is adjustable, there will be two or three small screws on the outer ring. In some models, the diaphragm is fixed to the condenser, so any alignment would be done with the entire unit.  To check the alignment of the iris diaphragm, follow the steps below. 1 . Remove any samples, filters, or other additional inserts such as annulars. 2. Switch on the light source and adjust the brightness to a level that is comfortable to look at down the eyepieces. 3. Close the iris diaphragm to the full extent. 4. Looking down the eyepieces, open the iris a small amount. There should be a small circle of light visible. 5. The small circle of light should be in the exact centre of the field of view. 6. Open the iris slowly to it’s full extent. The circle of light should stay centred the entire time, regardless how open or closed the diaphragm is.

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7. If the circle of light does not stay centred in the field of view as the iris is opened and closed, the diaphragm needs to be centred. To centre the diaphragm, follow the steps below. 1. Looking down the eyepieces, open the iris a small amount. There should be a small circle of light visible; this indicates the location of the aperture. 2. While looking down the eyepieces, use the adjustment screws on the diaphragm to manoeuvre the aperture until it is in the exact  centre of the field of view. 3. Once centred, slowly open the iris diaphragm a small amount  more. It should remain centred in the field of view the entire time that it is being opened. If it does not, then repeat the previous steps 1 and 2 with the diaphragm open to different extents. The aperture should always be centred no matter how open or closed the diaphragm is.

10.2 The Condenser After the light has passed through the diaphragm, it is focused onto the sample by the condenser. As the name would suggest, the role of the condenser is to take the field of light and to condense it into a focused point that lands on the sample. This triangular beam of light is known as the ‘light cone’ (or ‘cone of light’). By concentrating the light to a fine point, the light becomes more powerful and provides better illumination for the sample (Fig. 10.5). The condenser uses a high magnification lens to bring the rays of light into a fine point. The advantage of having a condenser is that the light will be sharper and it makes the most of the available light. The most common type of condenser is called an Abbe condenser. It is adjustable in the Z-axis to match the requirements of the different objective lens magnifications. This is where the user will need to consult the numerical aperture of an objective. • The NA of the condenser should be equal to, or greater than, the NA of the objective being used. This is to ensure that the entire field of view of the objective is filled with light. If the NA of the condenser is less than the NA of the objective, then light will be lost around the edges of the field of view.

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Upper lens

Iris diaphragm

Lower lens

Fig. 10.5  Cross-section showing the two lenses inside the condenser and the position of the iris diaphragm

10.2.1 How to Focus the Condenser For the condenser to accurately illuminate the sample, the focal point of the light cone created by the condenser should land on the sample. Adjusting the condenser is very simple and should be part of good practice when starting an imaging session. 1 . Remove any samples, filters, or other additional inserts such as annulars. 2. The light source of the microscope should be set to a level that is comfortable to view without there being a sample in the light path. 3. Lower the condenser as far down as it will go. 4. Place a small object such as a dissecting needle or the tip of a pencil on the light source—this will be used to judge focus. 5. Looking down the eyepiece, start to slowly raise the condenser until the dissecting needle (or whatever object is being used) comes into focus. 6. The condenser is now focused. Small adjustments might need to be made for when working at higher magnifications.

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References 1. Robert Hooke and the English Renaissance (2005) Gracewing. ISBN 0 85244 587 3 2. Turner G (1981) Collecting antique microscopes. Christies South Kensington Collectors Series. ISBN 0289708826

11 Light Sources

There is a single light of science, and to brighten it anywhere is to brighten it everywhere Isaac Asimov

Abstract  Good lighting is an essential component of good microscopy. There are numerous different light sources available for microscopy, and this can make it a confusing topic. For the level of microscopy in this book, knowledge of the different bulbs is useful but not essential. As a beginner microscoper, the choice of light source will likely have been made for you. The hobbyist or student user will most likely have an LED light source, unless they have an older model. In this chapter, we will cover the different types of light source and the different bulbs that are available.

The light source is a vitally important part of the imaging system. The quality of the image captured will be directly affected by the type, quality and characteristics of the light that is chosen. For the beginner or hobbyist user, there are not many different types of light source to choose from, but this is an advantage, as it gives the user one less component to be concerned with. Hobby and student microscopes will usually have a built-in light source, and modern styles are moving rapidly towards LED (also known as solid-state light) [1].

© Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_11

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For mid-range to larger systems, external light sources are commonly used. Older systems will often have external light sources or have larger light sources that are fixed to the back or side of the microscope. If the user is considering buying a second-hand microscope, this is something to be aware of. Historically, lighting for microscopes was done using mirrors that reflected light from the environment to the sample. Early methods of increasing light levels involved using a glass globe filled with water to focus the beam of light either into the mirror or directly onto the sample. In the nineteenth century, this evolved into paraffin lamps that bounced light off a concave reflector made of plaster of Paris to create white light [2]. Perception plays a role in choosing which light source to use. For example, modern microscope users are used to brightly lit samples, and find mirror lighting too dark to work with. However, look at Hooke’s book Micrographia and you will see what can be achieved with ambient lighting. If users are new to imaging, they will likely be attracted to the crisp brightness of LEDs. People who are experienced in imaging or have used microscopy for a large part of their career will often have a preferred type of light. Experienced pathologists, for example, are often not keen on LED lighting as they consider it too bright and white, and report that it washes out the colour of the sample.

11.1 Different Types of Light Source Light sources are classified as either internal or external and then by the type of bulb they use. The microscope model being used will usually determine whether the light source is internal or external.

11.1.1 Internal Light Source Internal light sources are built into the base of the microscope. They are generally not interchangeable or removable. The advantage of internal light sources is that the imaging system is all one piece. This makes it easier to store and use, eliminates the chance of losing parts, and lowers the desktop footprint of the microscope. The disadvantage is that it can be harder to replace bulbs or install a new light source if the previous one fails. If using a microscope that is going to be moved around a lot (e.g. taken in and out of storage), this is the best option to go for. Most student, hobbyist, and beginner microscopes will have a built-in light source.

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11.1.2 External Light Source External light sources contain the bulb in a small box, and the light from the bulb is fed to the microscope by a light cable. An external light source will also have its own power source. The advantage of an external light source is that it is easier to change the bulb or even swap out the entire light source if there is a defect or technical issue. If necessary, they are also interchangeable between machines  (depending on the model of the microscope and light source, of course). Systems with external light sources are best suited to an environment where imaging is being done in a designated area i.e. the systems does not need to be moved. The disadvantage of having an external light source is that it takes up more space than a built-in one, and it is an extra machine that can have its own issues. Safety: External light sources are safe to use, but the user must exercise caution when changing the bulb. Microscope bulbs get extremely hot, so the user must ensure the bulb has cooled before attempting to change it. Breaking the glass and releasing the chemicals of the bulb is another potential hazard. See Sect. 15.11 on how to safely change a bulb. Correct alignment of the bulb and optic cable is essential when using an external light source. If they are misaligned, then the light will appear darker on one side of the field of view. For more on this, and how to resolve it, see Sect. 15.6.

11.2 Different Types of Bulb Different types of bulbs have different characteristics, and these are important to know so that the user can make the right choice for imaging. The different gases and elements involved in manufacturing bulbs result in variations in the colour and temperature of light. The temperature of light is how warm or cool the light appears to be. Warmer light is more yellow in colour and cooler light is bluer. Consistency is key and over the duration of a study, the user must ensure they are using the same light source for imaging.

11.2.1 LED (Light-Emitting Diodes) More and more, contemporary designs of microscope are using built-in LED lighting. Their small size, reliable light, and economical running costs are making them the first choice for many microscope users.

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Cost: LEDs themselves are quite cheap to buy individually, but for microscopy, LED arrays are used. A LED array is a panel (known as a PCB or Printed Circuit Board) with a number of LEDs that work  together. Purchasing a LED array is more expensive than the traditional bulb light source, but over the lifetime of the microscope, they will not need to be replaced as often as a traditional bulb would. Over the life of the machine, they do  work out cheaper. Another advantage of the LED array is that, if one LED in the array dies, normal imaging can usually continue until the array can be replaced. Efficiency: LEDs are very energy efficient, and this makes the running of the microscope more economical and environmentally friendly [1]. Colour of light: The light from white LEDs is very pure. White LEDs will give a bright and crisp light to the sample. If the user has normally used halogen or another type of lighting and then moves to LED, they might find that LED light is too white and that colours in the sample appear washed out. The colours in the sample lit by LED will be the truer colour, but if the user is used to a warmer light, then it will take a little getting used to. Use: LEDs are very straightforward to use—simply switch them on and start imaging. LEDs do not require time to warm up, so imaging can start straight away. They also cool down quickly and are not hot to touch, eliminating burns as a hazard. As they are small, they are also easy to fit into more compact designs such as ‘lab on a chip’ applications [3]. Reliability: LEDs are a very reliable technology. They are easy to use and difficult to break. LEDs are very robust and can handle a certain amount of roughhousing. They are not as sensitive to temperature as traditional bulbs, so ambient conditions will not influence the light or colour of light being emitted. The only drawback of LEDs—other than their initial expense—is that LEDs tend to die suddenly. The user usually won’t have any warning that the LEDs are reaching the end of their lives. Heat: LEDs are non-heating. This is important for if the user is imaging live cells or organisms, as the heat from the light source can kill the sample. A sufficient heat sink should be part of the light source and LEDs will often sit on a ceramic plate to allow for efficient cooling [3]. Life: LEDs are extremally long-lived, and the user can expect to get about 10,000 hours of activity from them. LEDs can die quite suddenly, as opposed to traditional bulbs which will dull before they die. If the LED is one of an array, then this will not stop imaging, as the other bulbs should be bright enough to compensate. It could affect the appearance of how even the light is across the field of view though. Light loss: LEDs can be directed quite accurately, meaning less light is lost from the system. Many arrays include a parabolic mirror to reflect as much light from the LEDs as possible [3].

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Safe: LEDs, if broken, do not shatter the way a glass bulb does. They do not contain hazardous materials such as mercury. Adjustable: The level of brightness can be adjusted at the light source by simply changing the current [3]. This means there is less requirement for adjusting the diaphragm.

11.2.2 Incandescent Bulbs Incandescent bulbs are the traditional source of electric light for microscopy. There are a range of different bulbs that are used, and the most common ones are compared below. Incandescent bulbs are usually used with an external light source. Some smaller and older models of microscope will have a lamp housing that attaches to the rear or side of the microscope that houses the bulb. The general rules for using and handling the different types of incandescent bulbs are mostly the same across the different types. Consumable: Bulbs are considered a consumable in microscopy. The lifetime of the bulb will vary according to the type of the bulb, regularity and frequency of usage, how often the bulb is power cycled (turned off and on), and the ambient environment. Incandescent bulbs are usually not too expensive to purchase, as they are relatively cheap and safe to manufacture [4]. It is advisable to have one or two spares in stock in a busy lab. It is important to have the correct bulb for the light source, as incorrect current or voltage will negatively impact the colour being emitted and might blow the bulb. Efficiency: In comparison to LED lighting, incandescent bulbs are considered energy inefficient, as about 98% of the energy input becomes heat as opposed to light. Incandescent bulbs need to be allowed to  warm up for approximately 20 min [4] before imaging can start. This means that the system cannot just be switched on and off for quick imaging. If the microscope is in a busy lab and will be needed frequently, then the system is best left on during working hours. If there is a power cut while the light source is on, the bulb must be allowed to fully cool down before being switched on again. This cool down time before restarting can means losing imaging time.  Some systems have an  instant restrike setting that allows for the bulb to be restarted again immediately. Heat: Incandescent bulbs get very hot during use. They are contained within the light source casing, so the risk of burns to the user  is reduced. However, this means that quick bulb changes cannot be done. Extreme cau-

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tion must be taken when attempting to change the bulb or troubleshoot any issues if the system has been on. The light can also heat the sample and this should be remembered when working with live samples. See Chap. 12 for more about working with live samples. Safety: Handling incandescent bulbs must be done conscientiously. Gloves should always be worn when handling the bulb. If the bulb is handled with bare hands, oils from the skin are transferred onto the glass. When the bulb gets hot, the oil from the skin heats up and burns black marks onto the glass. The bulbs are, of course, made of glass and the user must be gentle when handling them. Faulty bulbs, or bulbs coming towards the end of their lives, can explode inside the light source. Metal-halide bulbs are particularly known for this, although through good bulb management, this should not be a common occurrence. Broken glass is a hazard, and some bulbs contain toxic chemicals such as mercury. Light loss: As incandescent bulbs are contained within an external light source, the light from the bulb is delivered to the microscope via a fibre-optic light cable. Once the light source and the fibre-optic cable are fitted correctly, there should be little loss of light. Fibre-optic cables should not be bent sharply, as any kinks in the cable will prevent light from passing through it correctly. End of life: One of the advantages of incandescent bulbs over LEDs is that they will give indications when there is a problem or when they are reaching the end of their lives. Most types of incandescent bulbs will start to flicker as they come to the end of their lives, and the user will see the bulb struggling to light up fully. This can be a useful trait as it indicates when the bulb needs to be changed. Another good indicator that the bulb needs to be changed is that the ends of the bulb become blackened, and the general colour of light they are emitting will start to become more yellow and dull. With proper bulb management, these indicators will give the user ample opportunity to replace the bulb and prevent any interruption to normal use of the imaging system.

11.2.3 Halogen Halogen lighting has been a popular option for microscopy for many years, and many systems still use it today.  • Halogen light is white but starts to yellow as the bulb ages. As halogen has been a popular choice in the past, many older pathologists prefer it and feel that it gives a richer colour to the sample.

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• The light of a halogen bulb has a steady output, and the bulb does not blacken, therefore maintaining even transparency across the bulb [5]. • Halogen bulbs require a high temperature to be bright, so they do not work very well in cold environments. The current being run through the bulb can also impact the colour of light being emitted [3], so it is important that the power source for the light is functioning correctly. • A blue filter can be used to adjust the colour of the halogen light, to give it a more ‘daylight’ tone [3]. • Under optimal conditions a halogen bulb can run for approximately 3600 hours.

11.2.4 Mercury • Mercury bulbs are efficient to run but there are restrictions upon how the bulbs can be disposed of due to the hazardous materials involved [4]. Mercury bulbs should be treated as a biohazard. • Under optimal conditions, mercury light is a brilliant white. There can be a blue tint, but colour-corrected bulbs coated with a layer of phosphor to whiten the light can be bought. • Mercury bulbs retain their colour of light throughout the majority of their life. • Under optimal conditions, a mercury bulb can run for approximately 200–300 hours.

11.2.5 Metal Halide • Metal halides are similar to mercury bulbs, but they are a more modern design with better energy efficiency and better colour rendition. • Metal-halide bulbs, once fully warmed up, produce brilliant white light. During warming up (or if there is not enough power reaching the bulb), the light can appear slightly blue or red, depending on the variety. • Under optimal conditions, a metal-halide bulb can run for approximately 2000 hours.

11.2.6 Fluorescent • Fluorescent bulbs come in a range of sizes and shapes. • Fluorescent bulbs can be more expensive to buy than halogen or mercury, but this can be offset by their efficiency.

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• Fluorescent bulbs can experience ‘cold spots’, where the bulb heats and therefore illuminates unevenly. This will result in uneven light across the field of view. • Fluorescent bulbs come in warm, neutral, cool, and daylight white. Daylight bulbs are slightly blue in colour. The warmer light is often easier to use for viewing, it is more comfortable for the viewers eyes, and therefore the user can image comfortably for longer. This will however affect the perceived colour of the sample. • Fluorescent bulbs contain mercury and so are classed as hazardous. When the mercury has been used up in the bulb, it will be seen to become pink. The lifetime of fluorescent bulbs varies depending on the type.

11.3 White Balance If using a digital system, it will sometimes be necessary to ‘correct’ the colour of light being emitted. This is known as performing a white balance.  The colour of light can change as the bulb ages and this can often be compensated for by adjusting gain levels. Consult the system manual or contact the systems customer support team for guidance on the correct gain levels for the system. Gain levels will differ between systems and different light sources, so there is not one set of values that will work on all systems (Fig. 11.1). Performing a white balance means telling the imaging software what white looks life. From this information, the digital system can make adjustments to give truer colours to the image. Performing a white balance on a digital system will usually be a very simple process. Usually the user will place a blank glass slide on the microscope stage (or leave it empty) and select the white balance option in the imaging software. I have yet to see a digital system that can warn the user that a white balance is needed, and this is an example of where

Fig. 11.1  Performing a white balance will correct the colour of the image on digital systems. The correct lighting in the centre image. The image on the left is too blue, and the image on the right is too yellow

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the  users knowledge of the sample is essential. If the colour looks wrong, it is wrong. 

References 1. Schubert E, Kyu Kim J  (2005) Solid-state light sources getting smart. Science 308(5726):1274–1278 2. Fiddian (1869) A new lamp for microscopic purposes. Mon Micros J:56–58 3. Wessels J, Pliquett U, Wouters F (2012) Light-emitting diodes in modern microscopy – from David to Goliath? Cytometry 81A(3):188–197 4. MacIsacc D, Kanner G, Anderson G (1999) Basic physics of the incandescent lamp. Phys Teach 37:520–525 5. Handbook of biological confocal microscopy (1990) Plenum Press. ISBN-13: 978-1-4615-7135-3

12 Choosing the Right Technique

Sound science must be our guide to choosing which problems to tackle and how to approach them Frank Luntz

Abstract  Brightfield microscopy is the technique that most people think of when they think of microscopy. It is indeed the most commonly used and usually the first technique that you will learn. It is limited, however, to samples that are thin and flat. You need not be restricted, and there are a range of techniques that are easy to use and accessible to the beginner. I have included the most common techniques here—brightfield, darkfield, phase contrast, Z-stacking, and stereomicroscopy. If you can master these five techniques, you will be very well equipped to deal with most kinds of sample. We will see what each technique looks like, what sample it is best suited for, the tech specs for the microscope, and the sample requirements.

When it comes to choosing what kind of equipment to use, what imaging technique is best, and what kind of images to capture, the first question should always be “what result am I looking for?” By knowing what end result is required and working backwards from that, the user will find choosing the optimal microscopy technique much easier.

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Important Note for Working with Live Samples • When working with live samples it is imperative for the user to remember the obvious—the sample is alive! Live samples can be cells in suspension being used for drug trials, they could be amoebas, spirogyra filaments, or even larger micro-organisms such as nymphs and larvae. Live samples must always be treated with sympathy and consideration. • Consult previous studies and/or primary literature to find out the optimum conditions for the particular live sample. These conditions should be maintained as accurately as possible throughout imaging. • When viewing live samples, the light should not be left on for long periods of time. The light can cause distress and the heat might eventually  kill the sample. • Allow live samples to regularly rest by switching the lights off. • Live samples need space. They cannot go on a normal glass slide with coverslip, as this will crush them. Live samples will require cavity slides (see Fig. 13.1), a petri dish or a multi-well plate, depending on the sample and what work is being done. A cavity slide is a glass slide with a little well in the centre where live cells or organisms can be kept in water. The coverslip is placed over the well to contain the liquid. Do not fix the coverslip in place—the live sample should be returned to a proper container or it’s natural environment after imaging.

Different microcopy techniques produce vastly different results. Knowledge of the different techniques and the kind of results they produce, enables the user to get the most out of their sample. Preparation for imaging should begin with the user deciding what kind of results they need to obtain from their sample. What is the user actually looking for—external features, internal features, behaviour, etc? Is there a feature in particular that needs to be highlighted? Is the sample live or fixed? Does the sample need to be imaged once or many times? The best place for the user to start is to review what other researchers have done previously. The user should review the primary literature and refer to images of the sample that are similar to what they are  trying to achieve. However, the user should not be afraid to experiment with different techniques, if time and resources allow it. The techniques described below and in this book are only a small sample of the imaging techniques that are available in research today. The techniques covered here are light microscopy techniques, and they are techniques that are readily available to most users.

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Fig. 12.1  Bone tissue being imaged using brightfield. BF is the traditional technique that most people associate with microscopy

12.1 Brightfield 12.1.1 What Does It Look Like? Brightfield (BF) is the technique that most people think of when they think of microscopy. BF can be recognised by the background (field) being bright white and the sample being highly contrasted against it. The image is created by the light passing up through the sample and into the objective. Samples for BF are usually individual cells (such as smears or cytology) or thinly cut tissue samples (Fig. 12.1).

12.1.2 When to Choose Brightfield • BF gives the user structural information of the cell (membrane, cytoplasm, and nucleus), and structural and organisational information in tissue samples. The user will be able to see the overall shape of the cell and the organisation of the cells within the tissue. • BF is the most common technique used in pathology and histology. • BF is mostly used for imaging fixed cells and tissues. • If imaging live cells using BF, the user should use as low a light level as possible and image quickly to reduce stress on the live sample.

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12.1.3 Tech Specs • BF is a low-tech imaging technique and the equipment inexpensive and easy to use. • The light shines up through the sample and into the objective. • BF objectives are the standard objectives that the microscope will come with. • BF does not require a special light source. • The most common magnifications for BF imaging are 5×, 10×, 20×, 40×, 60×,* 63×*, and 100×*. *When imaging at the higher magnifications, oil and oil objectives will be required. Remember that samples imaged under oil must have a coverslip.

12.1.4 Sample Requirements • BF samples must be thinly spread cells (such as a blood smear) or thinly cut tissue. • BF is used primarily for pathology and histology—samples for diagnosis are usually reviewed at 20× or 40× magnification. • Fixed samples will be stained and cover-slipped. • Fixed samples for BF can usually be stored at room temperature in a slide folder or storage box. Note: Samples that are being imaged under BF will be subject to heat from the light. This is not an issue when the sample is fixed but live samples must be rested, as described above.

12.2 Darkfield 12.2.1 What Does It Look Like? Darkfield (DF) can be recognised by the background (field) being black and the sample being illuminated against the background. In BF, the sample is illuminated by direct light. In DF, the sample is illuminated with gentle, reflected light coming from the sides (Fig. 12.2).

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Fig. 12.2  Darkfield (left) compared to brightfield (right)

12.2.2 When to Choose Darkfield • DF imaging is used to look at live cells and small organisms such as water fleas or larvae. • DF is less damaging to live samples as the lower level of light means the sample does not get subjected to as much heat as in BF. • DF samples are unstained and relatively transparent. • DF is used for looking at the overall external structure of the sample. Some internal features will be visible depending on the size and opacity of the sample.

12.2.3 Tech Specs • Most BF microscopes can be converted for DF. A DF condenser is required. • DF objectives are larger in order to capture more light. A different objective head might be needed to accommodate the larger objective. • DF does not require a special light source, the technique is created by blocking the central rays of light from reaching the sample. • A DF condenser has a 'stop' placed below the condenser to block the central rays of the light source. This creates a hollow cone of light that shines up and around the sample, like a halo. The sample is illuminated by this light from the sides. The NA of the condenser should be higher than the NA of the objective during DF imaging.

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12.2.4 Sample Requirements • Samples for DF are usually live cells or small organisms. • Samples are unstained—this is an important advantage of DF as some cells and organisms are very sensitive to chemicals and cannot be stained.

12.3 Phase Contrast 12.3.1 What Does It Look Like? Phase contrast uses diffused light to gently illuminate the sample from the sides. The background and the sample will appear grey, with varying levels of contrast creating the detail in the image. The sample will have good internal contrast and may appear to have a slight halo of light around it (Fig. 12.3).

12.3.2 When to Choose Phase Contrast • This method is particularly suited to viewing live cells and single-celled organisms such as amoeba.  Like in DF, this technique does not require staining, so live samples are viewed in their natural condition and natural

Fig. 12.3  Bone cells imaged using phase contract

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behaviours can often be observed. DF and Phase Contrast are similar techniques—DF is better for looking at external features and Phase Contrast is better for looking at internal features. • Phase is used to view the internal detail of live samples. External features can also be seen but are mostly in silhouette and obscured by the halo. • This method is popular for observing  cells over time, as the gentle light does not stress the sample as much as in BF.

12.3.3 Tech Specs • A standard BF microscope can be converted for Phase Contrast (Phase) by adding Phase objectives, a phase plate, and a condenser annular. • A special attachment—the annular—is added to the condenser and a phase plate is added to the objective. These can usually be fitted to an existing BF microscope. The annular disc (below the condenser) is a solid disc with a transparent ring in it. The phase plate (above the objective) is a clear disc that has a ring marked onto it. Together, these rings diffract the light and give the soft illumination required for Phase. • Phase Contrast objectives are required. • Phase does not require a special light source, the technique is created by strategically blocking the light.

12.3.4 Sample Requirements • This technique is ideal for the examination of internal structures, and the  observation of the natural behaviour of live  cells and micro-organisms.

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12.4 Z-Stacking 12.4.1 What Does It Look Like? Z-stacking is a technique that captures images of the sample while moving up or down through the vertical axis. This is an image-capture technique that can be applied to many different microscopy techniques.

12.4.2 When to Choose Z-Stacking This technique is used to create a pseudo-3D image of the cell or tissue and can be used to gather depth and distribution information from the sample. For example, the arrangement of cells within a tissue, or the arrangement of mitochondria within a cell. This technique allows you to move vertically up and down through the sample.

12.4.3 Tech Specs Z-stacking can be done manually or done using automation. Both approaches involve imaging the sample in the Z axis. The method for Z-stacking is to focus the objective at the top of the sample, capturing an image, and then moving down the Z axis a few microns, capturing another image, moving down through the Z axis, and continuing this approach until the bottom of the sample is reached. The images are then merged to create a composite image that allows the user to navigate up and down through the sample. The requirement for the microscope is that it can hold a steady position in the Z axis and perform image capture. On digital systems, some softwares have a Z-stacking mode where the user can define the top and bottom of the sample, how many layers to capture, and how far apart each layer should be. The software then guides the microscope and composites the image.

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Consideration for the size of the image file is important. One Z-stacked image will be significantly larger than one single-plane image.

12.4.4 Sample Requirements • Z-stacking can be performed on most well-prepared fixed samples. While it can be done on live samples, if the sample moves during imaging the individual layers of the Z-stack will not align properly.

12.5 Stereomicroscopy 12.5.1 What Does It Look Like? Stereomicroscopy is used to give users who are working with larger samples some magnifying power, will still maintaining depth perception. See Sect. 6.4 for more on stereomicroscopes.

12.5.2 When to Choose Stereomicroscopy • This method is particularly suited for doing work such as dissections, soldering, or any work that is done on a small scale. • This method is popular across many different industries including biologists doing small dissections, jewellery and watchmakers, people soldering circuit boards, etc.

12.5.3 Tech Specs • Stereomicroscopy is a low-tech imaging technique and is quite easy for a user to learn. • Stereomicroscopes are monobjective, meaning they only use one objective to achieve different magnifications. They do not have interchangeable objectives. • Reflected light is used to view the sample.

12.5.4 Sample Requirements • There are no special requirements for samples that are being viewed under a stereomicroscope.

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• Larger live samples such as water organisms can be viewed in their container without the need for transferring them to a cavity slide. • No staining required. • There is no limit to sample size, other than what can fit under the objective.

13 Sample Prep

‘Of these there are multitudes, many of which I have observ’d through a Microscope, and find, that they do, for the most part, everyone afford exceeding pleasant and beautiful objects’ Robert Hooke, Micrographia

Abstract  When it comes to sample prep, the most important thing to remember is this—garbage in, garbage out. No matter how good the microscope and no matter how good the microscoper, a bad sample will always be bad. The effort in microscopy should not be in image editing; it should be in sample prep. I have kept this chapter slightly generic, as you could dedicate an entire book to sample prep techniques alone. The best advice for the microscoper is to (a) research your sample and how other scientists have prepared it and (b) experiment! Take your time with sample prep—it is the key to good microscopy.

Good sample preparation is the key to good microscopy—a high-quality image cannot be achieved from a poorly prepared sample. It is possible to purchase samples that have been expertly prepared (see Sect. 13.1 Prepared Slide Sets below), but one of the vital skills of the microscope user is the ability to correctly prepare samples. However, the new user must not be intimidated by the practice of sample prep. Let it be just another skill to learn and experiment with. Learning good sample prep takes time and practice, and the © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_13

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new user should embrace this time of experimentation. Try different cuts, different mounts, and different staining—it is all part of the learning experience. Samples for microscopy must meet certain criteria, depending on the particular sample and the technique being used. The requirements for samples will be based on how the light and the sample need to interact. For example, in brightfield, light must pass through the sample. Whereas in darkfield, the light bounces off the sample. The most common method of sample prep that the user will start with is sample prep for brightfield microscopy. The requirements for brightfield samples are: • • • • •

The sample must be thin. The sample must not vary in thickness across the slide. The sample must lie flat and without folds in the tissue. The staining should be moderate, not too light or not too dark. There should be no air bubbles or foreign bodies (dust, hair, etc.) under the coverslip.

Sample preparation should not be rushed. The extra few minutes it takes to make a well-prepared slide are worth the effort. Practice makes perfect, and in time, the user will become proficient in preparing a range of different samples.

13.1 Prepared Slide Sets For users who do not have the resources to do their own sample preparation, boxes of prepared slide sets can be purchased. These are boxes of samples that have been expertly prepared and are ready to use straight out of the box. They are usually sold by category, for example, ‘invertebrates’ or ‘botany’. Samples for brightfield microscopy do not expire (although they can degrade, but this takes a long time). Sets of prepared slides are not expensive and are great for use in science education and outreach, or for users who are starting to explore science and microscopy, but who do not have access to a lab.

13.2 Equipment for Preparing Samples 13.2.1 Glass Slides The first thing the user will need for sample prep is glass slides. Microscope slides are traditionally made from glass, but plastic ones are also available. There are pros and cons for both glass and plastic (see the table below).

13  Sample Prep  Slide type

Advantages

Glass slides • Better optical quality • Less prone to scratching Plastic • Less likely to break slides • Safer for children • Cheaper than glass

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Disadvantages • Heavy if storing many slides • Hazardous if broken • Lower optical quality • Easily scratched (scratches interrupt clear viewing)

The standard glass slide is 1 inch × 3 inch and about 1 mm thick. Larger 2 inch × 3 inch slides are also available. Glass slides are not expensive and boxes can be bought quite reasonably. It is quite affordable to buy good-quality glass slides. The trained eye will easily see the difference between poor-quality and good-quality slides. Cheaper glass slides (a) will have a higher level of impurity in the glass, which causes scatter and artefacts in the image, and (b) will have an uneven surface. This unevenness will cause the sample to be uneven, which will show up in the image as out-of-focus areas in the sample. It is good practice to use one slide per sample i.e. don’t put different samples on the one slide. In research and lab work this is the standard approach however, this can vary. Some slides will have multiple different samples in order to do comparison work. The size of sample that can be put on the slide will be limited by the size of the coverslip (Fig. 13.1).

Fig. 13.1  Left to right, a standard glass slide, a cavity slide (note the circular dip in the middle for the sample), and two different sizes of coverslip

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13.2.2 Coverslips The coverslip is an extremally thin piece of glass that goes on top of the sample. It acts as both a protective layer on top of the sample, and it also flattens the sample for better focusing. Coverslips vary in width, length, and thickness, and the size that is needed depends on the sample. The objective might also have a recommended size of coverslip that it works best with, and this will be noted on the objective lens. The coverslip needs to comfortably cover the entire area of the sample. The thickness of the coverslip will vary depending on how robust the sample is. If the user is working with a sample that is very thin, a thinner coverslip can be used (the thinnest is around 0.13 mm). If the sample is a bit thicker or uneven, a thicker coverslip will be required (the thickest coverslips are around 0.64 mm). If the user is just starting to learn sample prep, choose the thicker coverslips to practice with, as the thinnest coverslips break very easily. Note:  Coverslips can very easily stick together. The user must make sure that only one is being used at a time. If two coverslips are used—one on top of the other—it will cause focus issues. Like slides, coverslips are traditionally made of glass, but plastic coverslips or coverslip tape is also an option. Below are some of the advantages and disadvantages of glass, plastic, and tape. Coverslip tape is usually used in high-throughput labs. Glass coverslips are the standard choice and give the best clarity. However, the user should do their own research too—and maybe some experimentation—to make an informed decision for what is best for the sample and for their workflow. Coverslip type Glass Plastic

Tape

Advantage

Disadvantage

• High optical clarity • Not as easily broken as glass • Safer for children • Some optical plastics can be just as good as glass • Can be cut to the required size and shape • Does not break • Is not hazardous to handle

• Easily broken • Lower optical clarity than glass • Easily scratched (scratches interrupt clear viewing) • Lower optical clarity • Can be difficult to get it to lie flat and smooth (creases or trapped air bubbles will interrupt clear viewing)

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13.2.3 Stains Many biological tissues, when cut thinly for brightfield imaging, will not have a natural colour or contrast strong enough for the user to see. If the sample has a natural colour, then staining might not be necessary. If, however, the sample does not have enough natural contrast and colour, then a stain can be applied. Staining methods and technology range from the simple and straight-­ forward, to the complex and highly specialised. Different samples will require different staining and use different procedures. It will be part of the users good practice to do some research on their sample to find out what the standard staining process it. For the purpose of this book, the procedure for simple contrast staining will be discussed. Stains can be bought in two forms, pre-made or as a dry powder that needs to be mixed. Pre-made stains will be more expensive, but if only small amounts are required then they are the better choice. The advantage of pre-made stains is, of course, that the user does not have to mess around with measuring powders or liquids. The disadvantage is the cost, but again, this is relevant to how much stain the user will be using. Staining mix is often less expensive than premixed ones, which is the advantage for labs where large amounts of stain are going to be used. The disadvantage of mixes is that it takes practice to prepare them correctly. Stain that is not properly prepared will (a) not be absorbed correctly by the sample, and therefore not provide sufficient contrast, and (b) poorly mixed stain, where the powder has not been properly dissolved, leaves dark spots (like tiny grains of pepper) on the sample.

13.2.4 D  ry Mount/Wet Mount and Temporary/ Permanent Mount A dry mount is when a sample is placed onto the slide and the cover slip is applied without adding any liquid. This is used for samples such as an insect wing, pollen, or hair. A wet mount is when a small amount of liquid (often water) is added to the sample on the slide before the cover slip is applied. This is used for samples where hydration is important for correct structure or if imaging a live sample that needs to be in water. The surface tension of the water holds the cover slip in place. This method is known as a temporary mount. Samples for pathology go through a number of steps. After cutting and mounting onto the slide, they are fixed. This means that they are prepared in such a way that the structures are preserved. The coverslip is held in place

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using mounting medium. This is known as a permanent mount and the slides last for a long time.

13.3 Health and Safety • Stains are chemicals and should be treated with respect. The user should wear gloves, any open wounds or cuts should be covered, and hands should be washed before and after the sample preparation procedure. • Do not do sample preparation in the same area that food is going to be prepared. • Keep all chemicals out of reach of children, and follow the usage and storage instructions that came with the stains. • If using glass slides and coverslips, the user must demonstrate common sense. Broken glass is dangerous. Broken coverslips produce very fine shards of glass that can easily pierce the skin. If slides or coverslips become stuck together, the user can try to gently pry or slide them apart, but they should not be forced apart. If the slides or coverslips cannot be easily separated, then they should not be used.

13.4 S  imple Thin-Prep Method for Brightfield Microscopy The following is a very simple method for preparing a thin-prep sample for brightfield microscopy. This is the method that most users will start with and it is a good way to get used to handling the materials. When starting out, the user should be prepared to make several attempts at this before they have a successfully prepared sample. Do not rush, and remember—practice and patience makes perfect. Different samples will have different requirements for sample preparation, the steps outlined below are a general guideline. When preparing a sample for mounting onto a glass slide, the user will need the following items: • • • • •

One glass slide per sample being mounted. Coverslips, one per glass slide. Tools for cutting the sample and a clean, non-slip surface to cut them on. Tweezers or forceps for handling the sample and coverslip. The required stain.

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• Mounting medium (if being used). • PPE (personal protective equipment, such as gloves and eye guards). As part of good practice, all samples should be considered a biohazard and treated as such.

13.4.1 Cutting the Sample Samples in the lab are cut using a machine called a microtome, or very small samples can be cut on an ultramicrotome (Fig. 13.2). A microtome uses blades to cut a very thin slice from a larger piece of sample embedded in wax. This is called the block. Training is required to use a microtome correctly, so this technique will not be covered in detail here. 1. If preparing a tissue sample, the first step is to examine the sample. Identify the area of the sample that is going to be cut and mounted. What is the correct orientation of the sample? Some tissue structures will look quite different depending on the orientation that you cut them. The piece of the sample that is going to be mounted should be no larger than the area of the coverslip that you are using.

Fig. 13.2  The microtome uses a very sharp blade to cut thin sections of the sample. Special training is needed to use a microtome

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Fig. 13.3  The sample should fit comfortably in the centre of the slide and be smaller than the area of the coverslip

2. The sample is cut as thin as possible. In the lab, different types of microtome may be available. If not, a sharp blade and a steady hand are required for this stage. The blade cutting the sample must be very sharp to reduce structural damage to the tissues. Tissues can sometimes be peeled in layers (like the skin of an onion). 3. The sample is placed on the slide using a tweezers. Lower the tissue in a slow and steady manner to minimise any air bubbles, gaps, or folds in the tissue (Fig. 13.3).

13.4.2 Fixation Fixing a sample is done to (a) preserve the structures of the sample as they were at the time of preparation, and (b) to preserve the sample for long-term use and storage. There are different chemicals and methods that can be used for fixing a sample, and the user should do some research into what is best for their sample. The general idea is to replace the water in the sample with fixative.

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13.4.3 Staining If only preparing one or two slides, the stain can be directly applied onto the slide. If many slides are being prepared, a ‘bath’ of stain can be prepared for the slides to be dipped into. Different stains will have different preperation and application requirements, refer to the manufacturer’s instructions on how to prepare and apply the stain correctly. It is important to mix and apply the stain correctly to the sample. Over-­ staining (stain that is too dark or applied too liberally) will prevent the details of the sample being seen. Under-staining (stain that is too light or has not had enough applied) will not provide enough contrast to show (Fig. 13.4).

13.4.4 Coverslipping Applying the coverslip requires a steady hand and a little patience. The goal is to have the coverslip lying flat and flush against the glass slide, without any air bubbles or artefacts trapped underneath it. Some people choose to rest the slide on the work surface during coverslipping. Some prefer to hold the slide in one hand and apply the coverslip with the other. The user should choose the technique that is the most comfortable and the steadiest (Fig. 13.5). If not using mounting medium: 1. The sample is now positioned centrally on the slide, and the stain has been applied (if stain is being used). Ensure that gloves are clean, to not transfer stain residue onto other objects or surfaces. 2. Open the coverslip packet and carefully pick up one coverslip by the edges if using fingers or by the tip of a corner if using tweezers. Remember, coverslips are very delicate. Go slowly! 3. Hold the coverslip in the dominant hand, and hold the slide by the label area or by the edges, with the other hand. If resting the slide on the work surface, use the non-dominant hand to keep the slide steady by holding the label area. 4. Place one edge of the coverslip flat on the glass slide, beside the sample. Slowly lower the coverslip. As it is lowered, the stain and sample will start to smooth and flatten. 5. Gently press down on the coverslip to flatten the sample and to push out any excess stain. Be gentle! This is a prime time for the coverslip to break.

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Fig. 13.4  Staining should be moderate. Too light a stain will not provide any contrast to highlight features. Too dark a stain will obscure the features

Fig. 13.5  A line of mounting medium is applied along one side of the sample. This will be the starting position for the coverslip

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If using mounting medium: 1. Different mounting mediums will have different application requirements, so the user should refer to the manufacturer’s instructions on how to prepare and apply the mounting medium correctly. 2. The sample is now positioned centrally on the slide, and the stain has been applied (if stain is being used). Ensure that gloves are clean, to not transfer stain residue onto other objects or surfaces. 3. A line of mounting medium is applied along one side of the sample. This will be the starting position for the coverslip (Fig. 13.5). 4. Open the coverslip packet and carefully pick up one coverslip by the edges if using fingers or by the tip of a corner if using tweezers. Remember, coverslips are very delicate. Go slowly! 5. Hold the coverslip in the dominant hand, and hold the slide by the label area or by the edges, with the other hand. If resting the slide on the work surface, use the non-dominant hand to keep the slide steady by holding the label area. 6. Place one edge of the coverslip flat on the glass slide to the outside edge of the mounting medium. Slowly lower the coverslip. As it is lowered, the mounting medium, stain, and sample will start to smooth and flatten. The mounting medium should distribute evenly underneath the coverslip (Fig. 13.6).

Fig. 13.6  Slowly lower the coverslip into place. The goal is to avoid trapping any air bubbles and to end up with a smooth and flat sample

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7. Gently press down on the coverslip to flatten the sample and to push out any excess stain and mounting medium. Be gentle! This is a prime time for the coverslip to break. 8. The mounting medium should be left to dry fully before imaging commences. By going slowly, the user should hopefully avoid catching any air bubbles under the coverslip. If an air bubble has been trapped, the coverslip can be reapplied, but it is best to land the coverslip successfully on the first attempt. Tip: Clear nail varnish can be used to seal the edges of the coverslip. This adds an extra layer of security and stops samples from drying out. If you have used mounting medium, ensure it has dried fully before sealing the edges of the coverslip. I have known people to use clear nail varnish as the mounting medium too, and this can be a good option for users who do not have access to a lab.

14 Image Capture

Photography was the medium pre-eminently qualified to unite art and science. Photography was born in the years which ushered in the scientific age, an offspring of both science and art Berenice Abbott

Abstract  As much as possible I have stuck with traditional, manual microscopy in this book. Digital systems are wonderful, and microscopy is moving more and more towards digitalisation. Digital microscopy will require an entire book of its own, so I have not gone too far into it in this book. I have included image capture as it is now a common addition to traditional microscopes and a logical next step for the user who wants to advance their skills.

More and more, digitisation and automation are being utilised in the lab, and microscopy has been one of the greatest adopters of this. By simply adding a digital camera onto the microscope, images of the sample can be easily captured, viewed, and shared. Once the fascinating world of microscopy is discovered, many users want to share and capture the views they are enjoying. In research, image capture has become an essential stage of many workflows. By capturing images of the sample, the user can create a permanent record of their work and findings. Digital imaging has made collaborating easier too. Previously, if a researcher wanted a second opinion on a sample, they had to ship the original glass slides to their colleague. This not only cost time and money, but certain types of samples (particularly human or primate tissue) are © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_14

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restricted from being moved between countries. Digital images of a sample can be kept indefinitely, and the original slides can be stored away or disposed of. If the user wishes to capture images on the microscope, there are many options and something to suit every budget. Note: The quality of the image will be a combination of the quality of the microscope optics and the quality of the camera.

14.1 Mounted Camera Mounted cameras are a style of camera that is attached to the microscope, usually as a permanent fixture. They are the most common type of camera used in professional labs, and the starting cost is usually in the hundreds. There are two general styles: cameras that are c-mounted and modular cameras.

14.1.1 C-Mounted Cameras The most common style of camera is attached to the head of the microscope using a c-mount (Fig. 14.1).

Fig. 14.1  A c-mounted camera on a trinocular head (left). A standard c-mount will fit most cameras (right)

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• A c-mounted camera will require that the microscope have a trinocular head (i.e. three ports for the light to go, two for the eyepieces, and one for the camera). • Trinocular heads will have a shutter that directs the light to either the eyepieces or the camera. The shutter can be opened and closed manually using a lever or by using the microscope software, depending on the system. • Some shutters are designed to direct the light 100% to the eyepieces or 100% to the camera. Some are designed to split the light 50/50, so that the user can see the image through the eyepieces and camera at the same time.

14.1.2 Modular Camera The other style of mounted camera in a module that goes under the head and sits in the light path between the objective and the eyepieces (Fig. 14.2). • A c-mount is not required for this style of camera, so the microscope can have a binocular head. • These styles of camera are often brand specific, as they are moulded to fit the body of the microscope. • Adding and removing this style of camera take a little effort. The user will have to remove the head of the microscope to add or remove the camera. 

Fig. 14.2  The modular camera is the white box underneath the eyepiece head 

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14.1.3 Software Mounted cameras will be sold with imaging software that is used to display and capture the images. Many of the softwares will also include some level of image adjustment, such as format conversion, adjusting brightness and contrast, and basic annotating.

14.2 Eyepiece Camera An eyepiece camera is a small camera that is slotted into the tube of the existing eyepiece on the microscope. This is a popular choice of camera for those who are also interested in astronomy, as the eyepiece insert can also be used on a telescope (once it has the correct diameter eyepiece). Eyepiece cameras are a great choice for the hobbiest user who wants to start experimenting with imaging at a more serious level. They can be bought online for between €50 and €150. • There is no requirement for a c-mount. If the user already owns a microscope with a binocular head, it will not be necessary to upgrade it  to a trinocular head. • The microscope must have a removable eyepiece. Some models have the eyepieces moulded into place. • The eyepiece camera can be quickly and easily added or removed. It can also be used on different machines once the eyepiece diameter is correct.  • Eyepiece cameras are connected to a computer and the image is displayed and captured on a computer monitor or tablet. The user should check that the eyepiece software is compatible with the computer or tablet they are going to use. One limitation of the eyepiece camera is that the field of view of the camera might be slightly smaller than that of the microscope eyepieces. This will result in losing the edges of the image; however, this should not be significant enough to prevent good imaging. Note:  Regularly removing the eyepieces of the microscope increases the chance of dust getting inside the system. If imaging is going to be regularly done, consider a fixed camera such as a c-mounted one or a modular one. 

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14.2.1 Software Eyepiece cameras will come with a disc or download link for drivers for the camera. Some softwares will also include some level of image adjustment, such as format conversion, adjusting brightness and contrast, and basic annotating. If these extra functions are not included, there are many comprehensive imaging softwares available online that the user can use to edit and adjust the images.

14.3 Camera Phone Adapter Mount Camera phone adapter mounts are eyepiece attachments that allow a camera phone to display and capture the image. These are quick and easy to add and remove from the microscope and can be easily thrown into a drawer or backpack when not being used. Camera phone adapter mounts are very affordable—there are lots available online for less than €20, and they require no software to run. If the user is just getting started or are imaging for fun, a camera phone adaptor will do the job just fine. These are also a great idea for students studying science, who will be able to quickly capture images during practicals for use during studying or for including images in essays and presentations.

14.3.1 Software Camera phone mounts do not come with software as the image capture is done using the imaging software on the camera phone. Remember that this will also be using the memory on the phone, so make it part of good practice to transfer the images off the phone and onto a computer or cloud storage. Software for adjusting and editing images can be downloaded from the internet; just make sure that a compatible image format is being used during image capture.

14.4 C-Mount Adaptor and a Regular Camera Many models of camera have threading for changing lenses. If a compatible c-mount can be found that fits the camera, then the user can attach the camera directly to the microscope. The advantage of this is that if the user already

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has a good-quality camera it can be adapted for image capture on the microscope. This set-up can work out well if the user is already a photographer or astrophotographer. • This set-up will require a c-mount that fits both the microscope and camera. • The usual configuration will be attaching the camera to the c-mount on a trinocular head. However, there are also adapters for attaching the camera to the eyepieces. • Adding a camera to the microscope can result in a bulky machine. The user must ensure that the weight of the camera will not topple the microscope.

14.5 Imaging Software There are many great imaging softwares available for viewing and editing images. • Purchasable softwares such as Adobe Photoshop are user-friendly and there are endless tutorials available online to learn how to use them. These softwares also have dedicated support teams to help the user with any issues. • Freeware is  software that has been made by individuals, usually to meet their own specific needs, that they have chosen to share online. Again, there are lots of tutorials online that will show the user how to get the most out of these softwares. During my Masters degree many of us used ImageJ, which has a wide range of algorithms available for different purposes. However, do take time to explore the softwares available to you, to find the one that suits you best. As freeware is often written by a number of different people, getting support can be a bit tricky and it is often at the discretion of the individual who wrote the software or algorithm. Note: While there are many options available to edit and enhance the images, the goal should always be to produce and capture a good image that does not need editing.

14.6 Good Practice Points for Image Capture • It is good practice to keep imaging conditions as similar as possible throughout imaging. This is particularly important if images are going to be used for comparison studies. Variation in light conditions can have a huge impact on the images.

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• Use the same type of light source each time. If possible, use the same microscope too. • If using a digital system, use the same computer and monitor each time. Check that the brightness, contrast, and colour settings have not been changed. Changes in the monitor settings will cause the user to change the settings on the microscope to compensate.  • If the user wears glasses at the start of an imaging session, they should keep wearing them throughout. Different people also have different visual ability and different opinions on what constitutes a good image. If multiple people are going to be doing the imaging for a project, they should agree on settings that everyone can replicate. • Before starting an imaging session, or a new project, consider how many images are going to be taken and what size the images will roughly be. Ensure that there is sufficient memory available on the computer or that an external memory source is available.

14.7 What Is a Digital Image? 14.7.1 How Is a Digital Image Created? A digital image is created using a series of numerical values organised into a grid. By changing the values of these numbers we can perform all kinds of image manipulations, such as changing the  brightness and colour levels, increasing and decreasing contrast, identifying objects,  and—most importantly for science—extracting data. To capture a digital image, light information from the sample is converted into an electrical signal by a device called a CCD.

14.7.2 What Is a CCD? CCD stands for Charge-Coupled Device. It is the part of a digital camera that collects the light and converts it into a digital image. The CCD has replaced film in traditional  cameras, and it  looks and functions much  like a tiny solar panel. As light travels through the sample, the photons are disrupted and visual information about the sample is created. The photons then travel from the sample, through any filters that might be in the imaging system, and onwards to the CCD.

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Each photon that impacts the CCD creates a charge. The more photons that hit the CCD, the stronger the signal. Based on the concentration and patterns of the photons striking the CCD, the digital image is constructed.

14.7.3 Pixels A digital image is made of a grid and each square of the grid is called a pixel. Most people are familiar with the term pixels, as digital cameras and smartphone cameras are classified by the number of megapixels (MP, one MP equals a million pixels) they have, for example, ‘12 megapixels’. The higher the number of pixels in a digital image, the better the resolution. This means that edges of features in the image are smoother and colours blend better across the image. To create the image, a value is assigned to each pixel (these values were obtained from the photons striking the CCD). In greyscale (black and white) images, there is one value per pixel. In colour images, there are three values per pixel. 

14.7.4 Resolution in Digital Images Microscope resolution is defined as the ability to tell two close together objects apart—so, for example, being able to tell that an object that appears to be one ribosome, is really two—they are just very close together or over-lapping. Resolution in digital images is defined as the number of pixels in the image. A high resolution image has more pixels per area and, the more pixels there are in a digital image, the smaller each pixel is. The smaller each pixel is, the smoother the detail in the image. Low-resolution images have fewer pixels per area and the image appears blocky. Higher resolution means smoother edges around objects and having better fine detail in the image. However, higher resolution also means a larger file size. File size will also depend on the file format, but this will be discussed further on in the chapter. While the temptation will be to go as high resolution as possible, high resolution also increases imaging time. This not only increases the hours spent infront of the microscope, but also increases the amount of time that the sample spends being exposed to light. This can result in photobleaching (loss of colour) and phototoxicity (build up of toxins in the sample). There will be a trade-off between resolution, time spent capturing the image, and file size. Many cameras for life science microscopy are around 3MP or 5MP. While this could be considered low to medium resolution, it is perfectly adequate for capturing great quality images of cells and tissues. The reason for this is that the 3MP and 5MP cameras have fewer, larger cells on the CCD. The

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larger the cells on the CCD, the better they are for catching photons. This makes it easier for weak signals to be detected and the fine details of the sample are captured.

14.7.5 Greyscale Black and white images are more correctly known as greyscale images. Each of the pixels in a greyscale image is assigned a single value, from 0 to 255. A value of 0 is pure black and a value of 255 is pure white. Every value between 0 and 255 is a shade of grey. Black and white cameras are better at detecting contrast in the sample. They also produce smaller image files, as each pixel only has one value compared to colour images, which have more than three. Black and white cameras are mostly used for fluorescent imaging, as they are more sensitive to light. This means they are better at seeing faint signals being given off by the sample.

14.7.6 RGB Colour To create a colour image, each of the images’ pixels is assigned three values. One value defines the level of red, one defines the level of green, and one defines the level of blue in each individual pixel. All the colours in a digital image are a variation of levels of red, green, and blue—hence the name RGB. Each of the three values is between 0 and 255. If a pixel is pure red, for example, its RGB value will be 255, 0, 0. For yellow, which is a variation of red and green, the RGB value will be in the range of 255, 255, 0. These are oversimplified values of course, and most RGB values will show much more variation. Images for brightfield microscopy are usually captured in colour, as staining is used to add contrast to the sample and sample colour can be used for diagnosis. Particular stains are also used for highlighting particular proteins or features. Colour image files will be larger than greyscale files.

14.7.7 Bit Depth Bit depth defines how many shades of grey or colour are in each pixel. For example, if a digital image is 1-bit, then the pixel has 21 options for its level of

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black and white. Therefore, a 1-bit pixel can either be black or it can be white, and the image will be a simple mosaic of black and white pixels. In comparison, an image that is 8-bit (28) has 256 options (256 shades of grey) per pixel. This will further increase the amount of information per pixel and also further increase the file size. Higher bit depth will give a digital image a richer colour pallet, but it will also give a bigger file size.  For life science imaging, 8-bit or 10-bit is usually enough.

14.7.8 Compression In digital imaging, the general rule is the more information in the image, the better. However, there is a fine balance between enough and too much. To mitigate the large size of image files, compression can be applied. Different image formats have different levels of compression. The goal is to reduce the file size without losing too much information from the image. There are two types of compression—lossy and lossless. Lossy compression is, as the name suggests, a form of compression where the file size is reduced by reducing the amount of information in the image. As the image data is compressed, some of the pixels are amalgamated, and they cannot be separated back out again. This results in smaller files which helps save memory and helps software work faster with images. However, applying too high a level of lossy compression results in the image losing quality and becoming blocky (i.e. losing resolution). Lossless compression is an algorithm that can compress the data in the image without losing any of it.  So the file size is reduced without losing information.

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14.7.9 Image Formats Image format has a direct impact on the quality and size of the image file and often there is a trade-off between these two. Different image formats (file types) have different built-in compression algorithms. The two most common image formats used in microscopy (outside of the brand specific formats) are JPEG and TIFF. JPEG (Joint Photographic Experts Group)—JPEG is a lossy compression format. It is a good medium between quality and file size. JPEG is suitable for images that are going to be used in a presentation or essays and course work. TIFF (Tagged Image File Format)—TIFF files are a commonly used lossless format. The file will be large but will be high quality. Images for publication in journals or on posters will often look best if TIFF is used. If data analysis is required, TIFF images will contain a high amount of information. Some digital imaging companies have created their own image formats. For example, Leica has the SVS format and Zeiss has the CZI format. Some of these are cross-compatible with different softwares, and some will only work on the brand specific software. This is something to keep in mind when purchasing equipment. It is useful for the user to know about the different image formats they are using and whether they are lossy or lossless. The image format will directly affect the quality of the images and whether they can be used for publishing or data analysis.  There are many more image format types than what is described above. It is advisable that the user check the formats available in the imaging software being used.

15 Troubleshooting

Above all, don’t fear the difficult moments. The best comes from them Rita Levi-Montalcini

Abstract  The best email I ever got about a malfunctioning imaging system said that the microscope was ‘making the Star Wars noise’. Your ability to troubleshoot issues will make you invaluable in the lab. People struggle to think clearly when they are stressed or frustrated, and again, this is where your strong knowledge of the basics will save the day. In this chapter, I have covered some of the most common issues that people come across. Pro tip—don’t ask people if the microscope is plugged in. They will just say ‘of course it is’, without checking. Ask them to check if the cable is loose or damaged at the plug end.

Troubleshooting can be a frustrating process, especially when the user is stressed or in a rush. However, narrowing down issues on an imaging system can be surprisingly easy once there is a basic knowledge of how the system works. This is one of the many reasons why knowing the basics of microscopy can give a user a huge advantage. Some of the troubleshooting steps below are glaringly obvious but, when under pressure and stress, it is very easy to overlook something simple. In the event of not being able to obtain an image or the image being poor quality, the user should go through each step one by one, in the following order. © Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2_15

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Note: Many microscope users do not take the time to review the system they are using before they start imaging. As a pre-emptive measure, the user should check the settings on the system before starting an imaging session. Some setting changes are not very apparent, and halfway through an imaging session is not the time to suddenly realise that the images are in the wrong file format or that the calibration on the image is wrong. Safety for the user and for the imaging system is paramount when conducting troubleshooting. The user should make sure that there are no slides on the stage (or in the autoloader if the system has one). As with any electrical appliance, the user should ensure their hands are dry and that they do not tamper with any damaged electrical connections.

Tip: • The best advice for anyone troubleshooting an imaging system is this—follow the light path. Start at the light source and follow it all the way through the diaphragm, condenser, stage, sample, objective, and up into the eyepiece or camera. This will unveil most issues or at least narrow down the possible causes.

15.1 A Word of Warning • The troubleshooting steps below are for a system that does not use a laser as its light source. Do not tamper with a laser light source in any way. Call a trained engineer. • Beware of moving parts manually on an automated system. Never force a part of the machine to move. If something is meant to be movable (or removable), it will move easily. • Remember that imaging systems can cost from a few hundred to tens of thousands of euro. If in doubt, do not touch! Seek help from an experienced technician.

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15.2 P  roblem: There Is No Light Visible in the Eyepieces, or the Light Is Dull or Uneven 15.2.1 Power This is a classic and will happen more often than anyone will ever care to admit. It may seem like a silly thing to check, but people will often unplug machinery to charge phones or plug in laptops and can forget to plug the microscope back in. The previous user might have switched the plug sockets off at the wall. If power cables on the microscope are compatible with another machine, the power cable might have been taken. Start at the power source

• Is the system plugged in? • Is the plug switched on at the wall? • Is the power cable securely seated into the machine? • Is the on switch turned to ‘on’? • Are the power lights coming on?

15.2.2 Light Source Correct lighting is essential for a good-quality image. Different samples have different light requirements, so it is very common on a shared system for other users to have made adjustments to diaphragms, lights levels, etc. Much information can be gleaned from the light coming through (or not coming through) the system. If the light levels are bright at the source but dull at the eyepiece, then there is likely something blocking the light path. If there are aberrations, flair, or coloured edges being seen, then there is an issue with the lenses. Being able to interpret the light in an imaging system is a useful skill. Type of light source Mirror

Check

Fix

• Is the mirror facing a good • Rotate the mirror and look for its reflection under the stage. Use light source? Is it receiving that as a guide to move the mirror enough light? • Is the mirror clean and able into the correct position • Increase the ambient light levels to reflect light? (continued)

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Type of light source Built-in light in the base of the microscope

External light source (a box beside the imaging system, which is connected to the microscope by a light fibre cable) Note: If the light source is a laser, do not touch it. Get a technician. Remember, a light source that has been switched on will be hot!

Check

Fix

• Is there any light coming out? • If no, is there a dial that changes the light level? Try adjusting this to see if it makes a difference • External light sources are powered independently. Repeat the power checks in step one for the light source. Most models have a fan that will start spinning once the light source has been switched on • Is the bulb in the light source lighting up? Remove or loosen the light fibre cable from the external light source and check. The light should be quite strong • Is the light reaching the microscope? Check that the microscope end of the light fibre cable is properly seated and secure. Remove the cable from the microscope end; is there light coming through it? The light should be quite strong

• If there is no light coming from the base (this will be clear to see) and the dial for adjusting light levels has been checked. If there is still no light, then the bulb probably needs to be replaced • How long a bulb will last in an external light source will depend on many factors—what type it is, how old the bulb is, and how much it has been used, if it is being switched on and off regularly or left on? The environmental conditions of the room can impact bulb life, cold conditions will shorten the bulb life. Lifetimes can vary greatly • External light sources aren’t completely light proof. There should be a small amount of light leaking out around the small gaps where the light fibre cable leaves the box • The light fibre cable itself can become bent and block light from travelling through it correctly. Remove the cable from the microscope end; there should be a strong light coming through. Make sure there are no sharp bends or kinks in the light cable. A damaged light fibre cable will need to be replaced

15.2.3 Diaphragm and Condensers Once it has been confirmed that the system is powered on and that light is reaching the system, the user will now work through the individual components of the microscope. Start at the light source and work towards the eyepieces or CCD.

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Fix

Is the diaphragm open? (Fig. 15.1)

• Look down the eyepieces and also directly at the diaphragm itself. You may need to look from underneath. Ensure that the diaphragm is fully open to allow the light through to the condenser • Ensure that the diaphragm is fully open to Is light coming out the top of the allow the light through to the condenser condenser? Look underneath the stage, where is the light falling— • Follow the steps in Sect. 10.2.1 to focus the on the underneath of the stage or condenser at the aperture in the stage where the light can reach the sample? Is it a small point of strong light or a larger area of soft light? • Some older microscopes have a blue filter Check for any filters positioned lens between the diaphragm and the between the diaphragm and the condenser. These are usually only found on condenser older systems and were used to tone down the light if it was coming through the system too brightly. Remove any filters and see how it changes the light coming through the eyepieces

Fig. 15.1  The edges of the iris diaphragm can be clearly seen in the field of view. The iris needs to be opened

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15.2.4 Stage When looking at the stage from above, the user should be able to clearly see the top lens on the condenser and if there is light from the condenser coming through. Is the stage blocking the light path?

Is the specimen holder (the part that moves the slide in the X and Y axis on the stage) blocking the light path?

• Look down from above; is light coming up through the stage where the slide should be? Can the entire area of the top condenser lens be seen? If no, the stage or condenser needs to be realigned so that the top lens can be seen and light can come through to the sample • As above

15.2.5 Objective After the power not being switched on, the other great classic is the missing objective. In larger labs, it is very common for people to swap objectives between machines. This results in there either being the wrong objective on the head, an incorrectly fitted objective, or no objective at all. Some microscopes will not have an objective in every available position on the objective head, so it is possible that there is an empty position on the turret above the sample. Check

Reason

Fix

Is there an objective in the light path? Is the objective properly in place?

• Light will only reach the eyepiece if it passes through an objective

Ensure that there is an objective positioned in the light path

Is the objective threaded correctly?

• Most turrets will click when Gently push the side of the objective—if it swings easily, it is the objective is in the not in place correctly. Turn it until correct position. It should it clicks or snaps into place take a little effort to move the objective out of position • When screwing an objective Unscrew the objective and reseat it. Once the objective is straight, into the turret, it is very position it in the light path. The easy for it to go in crooked. lens will be seen to become When in the correct brighter, as the light is now position, the objective hitting it correctly should be perfectly straight (continued)

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Check

Reason

Is it the correct type of objective?

Check that the correct type of • Air objectives will only objective is being used and, if work in air using oil, check that the RI of the • Water objectives will only oil matches that of the objective work in water • Oil objectives will only work in oil • The RI value on an oil objective must match the RI value of the oil being used Review Sect. 8.9 for working • If the objective and stage distances of the different are too far apart, the light magnification objective lenses. will not be captured by the The objective and stage must be objective and therefore at the correct distance cannot pass through the system properly

Are the stage and the objective too far apart? 

Fix

15.2.6 Shutter Shutters are found on systems that alternate between using the eyepieces and using a CCD/camera to display the image. The function of the shutter is to redirect the light path to either the eyepieces or the camera. Some systems have a 50/50 setting where the light can be directed to both the camera and eyepieces at the same time. On systems where it is 100% one or 100% the other, it is a common mistake that the user might be looking down the eyepieces but have the shutter in the CCD position or vice versa. Therefore, seeing nothing where they expect to see the image. Fix

Check

Reason

Is the shutter open/ closed?

If the shutter is manually controlled, there Alternate the shutter between the in and out will be a small handle located between position while looking the eyepieces/CCD and the objective. An through the easy way to find the shutter handle is to eyepieces (or at the follow the light path upwards from the monitor if using the objectives. The shutter will be located camera/CCD) somewhere between the objectives and the eyepiece or CCD/camera. It will be on the housing of the microscope, usually to one side. Different systems will have the ‘out’ position as the eyepieces, and some will have the ‘in’ position as the eyepieces. If the shutter is controlled by the computer, open and close it through the software. This will vary between brands, so the manual should be consulted

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15.2.7 Eyepieces There are a limited number of issues that can go wrong with eyepieces. The most common complaint with eyepieces is that they are dirty. This will happen naturally, as users will contact the eyepiece cups with their skin and oil from the skin, and moisturisers or makeup can build up on the eyepieces. Diopter eyepieces can sometimes cause trouble for users who are not familiar with how to focus them. If the eyepieces are removable, then they can sometimes go missing in busy labs or be swapped onto other machines. Check

Reason

Fix

Adjust the eyepieces outwards or The user should be looking inwards until each eye is looking down the centre of each down the centre of each eyepiece. If the eyepieces are eyepiece. The two fields of view too far apart or too close together, the user’s eyes cannot should overlap exactly—the user should not see two circles (See catch the light path correctly Sect. 7.3 for more on interpupillary distance.) • People who wear glasses (high Turn the eyepieces until they are Are the all the way down. Look through eyepieces at eyepoint) will adjust the the eyepieces and slowly adjust eyepieces differently than the correct them upwards until there is a someone who doesn’t (low height? clear field of view eyepoint) See Sect. 7.5.1 on how to adjust • Check if the eyepieces are diopter eyepieces diopter eyepieces Are the eyepieces too far apart?

15.2.8 Camera This step is for systems using a camera mounted onto the microscope. For eyepiece cameras, check the eyepieces and that the camera or camera phone is aimed directly down the eyepiece, and not at an angle (i.e. not looking at the inside of the tubing instead of at the sample). Check

Reason

Is the camera There will usually be a green light switched on? or similar indicator that the camera is switched on

Fix Check the power to the camera using Sect. 15.2.1. Cameras are often powered by the connection to the computer but some may have their own power supply (continued)

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Fix

Check the computer and Some cameras are powered by the computer or need the computer to monitor are switched on and awake. Check that the run user log-in has permission to access the software, that the software is available on the user log-in and that they have the correct permissions to access the software Check there is a cable Is the camera Most contemporary cameras will connecting the camera to connected to have a USB or a HDMI cable the computer connecting them to the computer. the Check that it is the correct Ensure that the correct cable is in computer? cable and that it is in the place. It is very common for cables correct port and securely to be swapped between machines seated or taken Shut down the whole system Many systems need to have the Were the and restart it. Start the camera started before the camera and camera first and then the computer is started. This is computer computer because, as the computer starts, it started in sends out a ‘hello’ to see what the correct other devices are connected. If the order? camera is turned off when the computer sends this message, the computer gets no reply from the camera and therefore doesn’t connect to it Remove any attached cables Is the camera If the camera is attached via a from the camera and c-mount, it is quite easy for it to positioned unscrew it. Make sure it is be sitting incorrectly correctly? straight and replace it on the c-mount, threading it carefully. It should be sitting perfectly straight on the microscope Check the Programmes list of Software Many cameras will be ‘plug and the computer for the play’, and the computer will software automatically search for the Open the software belonging required driver to the camera On some systems, the software Check the computer for might need to be manually available devices; the installed and set up camera should be listed • Is the software recognising the If the software is not camera? There will often be a recognising the camera, try drop-down menu to select the restarting the camera and model of camera from, or the computer, as described software will name the camera above. If that does not somewhere in the viewing work, try reinstalling the window software

Is the computer on?

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15.3 Problem: There Is a Shadow in the Image Dust and dirt in the system can lead to artefacts in the images. This is commonly seen as a shadow or a dark spot. Dust and dirt are usually easy to spot in the image but can be difficult to locate in the imaging system. Follow the troubleshooting steps below while looking down the eyepieces with the light source on (turn the light source down if necessary for comfortable viewing). Set up the microscope as normal and place a sample slide on the stage. Focus the sample as best as possible and work through the following steps. Fix

Check

Reason

How dark is the artefact?

The dirt is either on the condenser The shadow is very light. or the light source. Dirt and dust on the condenser will appear as Wipe the lenses of the condenser a very faint shadow in the and the light source with lens tissue background of the image The shadow is dark The dirt is either on the slide, the objective, or the eyepiece. Go to the next step The dirt stays in the same The dirt is either on the objective place or the eyepiece. Go to the next step The dirt moves when the The dirt is on the slide. Clean the slide moves slide No, the dirt is still there The dirt is on the eyepiece, clean the eyepiece (Sect. 7.7) Yes, the dirt is gone The dirt is on the objective, clean the objective (Sect. 8.14)

Move the slide— does the dirt move or stay in the same place? Rotate to a different objective—does the dirt disappear?

15.4 Problem: Cannot Focus During Air Imaging Check

Reason

The sample is If the objective can see, but not focus, there is coming into most likely an issue view, but it with distancing, or the won’t focus lens (or slide) has dirt or grease on it

Fix • Check that the slide is not upside down. The objective will not be able to focus through the slide the same way it can focus through the coverslip. Also check that the slide is clean • Check that the objective is clean and seated correctly • Check that the eyepieces are clean, correctly seated, and focused (continued)

15 Troubleshooting  Check

Reason

Focus the sample and The sample then watch the focuses objective. Is the initially, but objective drifting? does not stay in focus Focus the sample and watch the stage. Is the stage drifting?

If there are patches of The sample out-of-focus areas, will focus then it is most likely but some uneven tissue in the areas of the sample. It has either sample are been cut unevenly or out of focus the tissue is not lying flat on the slide

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Fix If the objective/stage cannot hold its position in the Z axis, then the sample will not stay in focus. Check the objective/stage controls are not loose or that oil has not gotten on the threads. If it is an older machine, the threads might be worn out. Loose fittings can be tightened but worn out threads cannot be fixed Better sample prep will solve uneven areas in the sample

15.5 Problem: Issues During Oil Imaging Check

Reason

Fix

Ensure oil has been applied and that the objective has correctly made contact with the oil. See Sect. 8.13 Check that the RI of the Has the correct oil been used?Is oil matches the RI of the the correct objective being used objective. and is it correctly fitted? Check that the correct objective is being used and that the objective is correctly seated Over time the heat from the light The immersion oil has run During an oil out and the oil objective thins the oil. As the user imaging session, is imaging dry. Reapply navigates around the sample, the image stopped the oil the oil gets spread around. Over focusing time, the oil wears out and needs to be replaced.  Clean the slide so that all After an oil imaging oil is gone. Ensure that session, the air oil did not get on the air objectives can’t objectives focus on the sample The image will not focus no matter how much the focus knobs are adjusted Oil has been applied, the objective has contacted the oil, but the image will not focus

An oil immersion lens will not work without oil. Has oil been applied? Has the objective contacted the oil?

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15.6 P  roblem: The Light Is Coming Through the System, But It Is Uneven The most common cause of this issue is that either the iris diaphragm, the condenser, or the light source has become misaligned. This will result in the light appearing to be over to one side of the field of view. Start with the condenser, then the diaphragm, and then the light source. Check that each one is centered, that the diaphragm is fully open, and the condensor focused. Centring is not normally an issue with a built-in light source, but the light fibre cables used in external light sources can come loose. Look through the eyepieces and open and close the diaphragm—are the leaves opening and closing smoothly and evenly? If the leaves are closing unevenly, you will see the circle of light moving over to the side as the leaves are opening and closing. They can usually be gently pulled into the correct position. Do not force the leaves or bend them. Check that the condenser is focused and centred correctly (See Sect. 10.2.1)

15.7 Choosing the Right Light Level There is no exact rule as to what the best level of light is for imaging—each sample is unique and will require different levels of contrast, brightness, and exposure. It is important to maintain the same light conditions for samples that are being used in time-lapse imaging and for samples that are going to be used for analysis or for any kind of comparative work (Figs.  15.2, 15.3, and 15.4). Advantages of bright light

• Easier to see • Easier for clear image capture • Can assist in imaging thicker samples or samples that have been stained too heavily • Required for higher magnification objectives Disadvantages of bright • If the light is too bright, it will wash out the sample light • If doing image capture and the light is too bright, the image will look overexposed Advantages of low light • Adds contrast • Safer for live samples • Better for very thin samples • Better for lower magnification objectives • Can be too dark to see with the eye or camera Disadvantages of low light • Loses colour

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Fig. 15.2  Overexposure of the sample results in washing out the colours

Fig. 15.3  Correct exposure of the sample shows the colours correctly and is comfortable for the user to observe

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Fig. 15.4  Underexposure results in an inability to see the features of the sample

15.8 Colour of the Sample Is Incorrect When imaging using a digital system, the colours of light can become altered due to aging bulbs or incorrect calibration. When using a traditional microscope, if the colour of the sample does not look right, it is more likely that light levels are the issue. Check and adjust the light source, diaphragm, and condenser. If using a digital camera, remember that the computer monitor setting can also affect the appearance of the image. Check the monitor settings for brightness, contrast, and saturation. 

15.9 The Wring Test Moisture can cause huge damage to a microscope, especially to the lenses. If using an alcohol wipe or any damp tissue/material, to clean any part of the microscope, always wring out the wipe/tissue/cloth first. The Wring Test—tightly wring the wipe/tissue/cloth. If any moisture drips or comes to the surface, it is too wet to apply to the microscope.

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15.10 C  entring an Objective on a Petrological Microscope It is essential that the objective on a petrological microscope is perfectly centred. This is an easy task to do but it does take a little bit of time and patience. Equipment needed: Prepared sample slide Pair of objective screws (these will have come with the microscope) 1 . Set up the microscope as normal and place the sample slide on the stage. 2. Check the alignment of the objective using the following method: (a) Looking down the eyepieces, choose a feature in the sample that stands out, i.e. something on the slide that will be easy to locate a second time. (b) Move the slide on the stage until the feature of interest (FOI) is in the centre of the field of view. (c) Start to rotate the stage and complete one entire rotation. If the objective is perfectly centred, then the FOI will remain in the centre of the field of view as the stage is being rotated. (d) If the FOI does not stay in the centre as the stage rotates, the objective needs to be centred. Proceed to step 3. 3. Ascertain which direction the objective needs to be moved in, using the following steps: (a) Centre the FOI in the field of view. (b) Rotate the stage and watch the direction the FOI moves in. Rotate the stage 360°—the FOI will do a loop and return back to the centre of the field of view where it began. (c) Rotate the stage again, but this time, stop when the FOI reaches it’s furthest point from the centre. (d) The objective needs to move from this furthest point, back towards the centre point. Go to step 4. 4. Take the pair of objective screws and insert them into the apertures above the objective. These will be located at the top of the objective, where it connects to the objective head. There will be one either side of the objective (two per objective).

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5. Looking down the eyepiece, with the FOI at it's furthest point from the centre of the field of view,  make tiny adjustments using the objective screws. It is best to adjust the objective screws a tiny bit in one direction at a time, i.e. up, then right, up, then right, etc. 6. Walk the FOI half way  back to the centre of the field of view using the screws. 7. Once the FOI is roughly half way back to the centre, stop. Move the slide on the stage the rest of the distance, so that the FOI is now all the way back in the centre. Rotate the stage 360°. Does the FOI stay perfectly in the centre? If yes, then the objective is correctly centred. If not, then repeat steps 3–6. It will usually take several attempts to get the objective perfectly centred. Be patient, and only make tiny adjustments.

15.11 H  ow to Change the Bulb in an External Light Source Warning! Never attempt to change a bulb until it has fully cooled down. The bulbs for microscopy get extremely hot. Always wear gloves when handling a new bulb. The oils from the skin can damage the bulb when it gets hot. 1. Switch off the light source and allow the bulb to cool fully. Unplug the power as an extra safety measure. 2. Locate the opening on the light source—this will usually be where the light cable enters the external light source. 3. Carefully open the light source. Keep the panel supported, as there may be wires connected to it. 4. Depending on the style of bulb, there may be one or two power connectors. Gently disconnect the old bulb and dispose of it accordingly. 5. Wearing gloves, take the new bulb and attach the connectors as they were attached to the old bulb. 6. Reseat the bulb and close up the light source. 7. Turn on the power and switch on the bulb. 8. If using a digital system, white balance and colour calibration may need to be performed.

Glossary

A Abbe condenser  the most common type of condenser for light microscopy. The condenser is located between the light source and the stage. It controls, focuses, and ensures even distribution of the light coming from the light source before it reaches the sample. The Abbe condenser is constructed of two lenses, one plano-convex and the other biconvex. Aberration  from the Latin ‘aberratio’ meaning ‘to stray’. In microscopy, an aberration is when the light rays do not align at the focus point correctly. This is caused by misalignment of the lenses or the light source in the microscope. The most common types of aberration in microscopy are chromatic aberrations or spherical aberrations. Absorption  absorption occurs when photon energy from light is absorbed by an object and converted into internal energy, such as heat. Achromatic (abbrev. Achro)  from the Greek ‘a-’ meaning ‘without’ and ‘khrōmatikos’ meaning ‘colour’. Achromatic means ‘without colour’. In microscopy, achromatic is used to describe an objective lens that has been designed to keep the colours of light combined as white light. Achromatic objectives correct for red and blue chromatic aberrations. Annuli  from the Latin ‘annularis’ meaning ‘ring’. A circular or ring-shaped attachment that is use to disperse light. See Phase annulus. Aperture  from the Latin ‘apertura’, meaning ‘opening’. The aperture is the hole through which the light passes. In microscopy, the hole in the diaphragm is an example of an aperture. In the human eye, the pupil is an aperture. Apochromatic (abbrev. Apo)  from the Greek ‘apo-’ meaning ‘away or off’ and ‘khrōmatikos’ meaning ‘colour’. In microscopy, apochromatic is used to describe an objective lens that has been designed to keep the colours of light combined as white light. Apochromatic objectives correct for red, blue and green chromatic aberrations. Artefact  something unexpected or unwanted in the image or field of view. An artefact can be something as simple as a speck of dust on the objective lens, which can be easily solved. They can also be something more permanent on the sample, such as an air bubble trapped under the coverslip, a fold in the tissue, or even granules of stain that have not been diluted properly.

© Springer Nature Switzerland AG 2019 D. Lawlor, Introduction to Light Microscopy, https://doi.org/10.1007/978-3-030-05393-2

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B Biconcave lens  from the Latin ‘bi-’ meaning ‘two’, ‘con-’ meaning ‘together’, and ‘cavus’ meaning ‘hollow’. A biconcave lens is a lens that curves inwards on both sides—making it thinner in the centre and thicker around the edges. Biconcave lenses bend the light rays outwards and magnify the image. Biconvex lens  from the Latin ‘bi-’ meaning ‘two’ and ‘convexus’ meaning ‘vaulted’ or ‘arched’. A biconvex lens is a lens that curves outwards on both sides. The lens is thicker in the centre than at the edges. A biconvex lens bends the rays of light inwards. At the point where all the light rays converge is the focal point. Binocular head  from the Latin ‘bini’ meaning ‘double’ and ‘oculus’ meaning ‘eye’. A binocular head on a microscope has two eyepieces, as opposed to a monocular head which has one, and a trinocular that has two eyepieces and a third pathway for a camera. The binocular head is the standard design for most modern systems. Binocular vision  from the Latin ‘bini’ meaning ‘double’ and ‘oculus’ meaning ‘eye’, vision is from the Latin ‘visio’ which means ‘to see’. Vertebrates and many invertebrates have two eyes, positioned slightly apart from each other on the head. Each eye perceives a slightly different angle of the field of view. The two images overlap in the brain to give the individual depth perception. Birefringence  from the Latin ‘bi-’ meaning ‘two’ and ‘refringere’ meaning ‘to break up’. A material that is birefringent has a different level of refractive index depending on the direction of light and the orientation of the material. This can be recognised as the material appearing to change colour as it is rotated. Birefringence is often used in geology to identify different types of mineral. BMP (Windows Bitmap)  a format built for handling images on the Windows Operating System. They are lossless, large files that are widely compatible across Windows. Brightfield (abbrev. BF)  a microscopy technique used for viewing cell and tissue samples. Brightfield is recognised by the background being white and the sample being stained to show physical features. Brightfield is usually the technique that people envision when they think of microscopy and is the first technique that a user will learn.

C C-mount  a threaded mount that can be used to attach a camera to the microscope. The c-mount is the male thread and the camera should have a female thread. CCD  see Charge-coupled device. Charge-coupled device (abbrev. CCD)  an instrument that detects photons using silicon cells. The CCD converts the charge of the photons into an electrical signal. This is then converted into the digital image. Chromatic aberration  from the Greek ‘khrōmatikos’ meaning ‘colour’ and the Latin ‘aberratio’ meaning ‘to stray’. A chromatic aberration is caused when the different colours of light have separated, causing a rainbow effect in the image. Individual colours of light will be seen around the edge of the field of view and around the edges of the sample. Colour temperature of light  the colour temperature of light describes how ‘warm’ or ‘cool’ a light appears to be. Warm light has a yellowish tint, whereas cold light has a blueish tint. Colour temperature is important for consistency during imaging, as different light sources have a different colour temperature. CMO  see Common main objective.

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Common main objective (abbrev. CMO)  a model of stereomicroscope where the light enters one large objective lens. CMO style stereomicroscopes have greater light gathering ability than the Greenough model. Compression  in digital imaging, compression is an algorithm that is applied to the data in the image to reduce the size of the image file. Different image file formats have different levels and types of compression. See also Lossy and Lossless compression. Concave lens  a lens that curves inwards at the centre. Concave lenses bend light ray’s outwards, spreading them apart and expanding the image. A concave lens is thinner in the centre and thicker around the edges. See also Biconcave lens and Plano-­concave lens. Cones  the photosensitive cells in the eye that detect colour. Cone cells have a faster reaction time to photostimulation than rod cells. This makes them better at tracking fast-moving objects. Cone cells are also better at seeing fine detail. There are three types of cone cell—one for blue light, one for green light, and one for red light. Humans are trichromat, which means they have three types of cone cell. There is a rare condition where a person (more prevalent in women) can be tetrachromat, which means they have four types of cone cells, allowing them to see the ultraviolet spectrum. Condenser  the condenser sits between the light source and the sample. In light microscopes, they are usually found under the stage, although in inverted microscopes, they are above the stage. The job of the condenser is to control the amount of light reaching the sample by adjusting the cone of light. This ensures the light is focused and evenly distributed over the field of view. Cone of light  used to describe the path of light as it moves from being a broader area of diffuse light, to a focused point of concentrated light, and vice versa. Convex lens  a lens that curves outwards in the centre. Convex lenses bend light rays inwards, bringing them together and focusing the image. See also Biconvex lens and Plano-convex lens. Course focus knob  the dial used to make large movements of the stage or objective (depending on the model of microscope) in the Z axis for focusing. Course focus is done while watching the stage, and not looking down the eyepiece. See also Fine focus knob.

D Darkfield (abbrev. DF)  a microscopy technique where the sample is illuminated from the sides, by a hollow cone of light. Darkfield can be recognised by the background being dark and the sample being bright but with no halo. DF is a good technique for imaging live samples or for looking at external features. Diaphragm  from the Greek ‘di’ meaning ‘through or apart’ and ‘phragma’ meaning ‘fence’. The diaphragm is also known as the iris. Like the iris in the eye, the iris in the microscope is opened and closed to control the amount of light entering the condenser and reaching the sample. The diaphragm sits between the light source and the condenser. Digital zoom  magnification that is achieved using software. Digital zoom is artificial and the image can become quite pixelated at high zoom levels, so it is really only effective for making small changes. See Optical zoom Diopter eyepiece  from the Greek ‘di’ meaning ‘through’ and ‘optos’ meaning ‘visible’. A diopter eyepiece is an eyepiece that allows the user to adjust the focus of each eyepiece individually. In many people, the visual ability of each eye is slightly different. The diopter eyepiece is used to accommodate this difference. They are particularly useful for users who have astigmatism. Diopter eyepieces are also commonly found on binoculars. Dissecting microscope  see Stereomicroscope.

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E Electron  a subatomic particle with a negative charge. Emission  in optics, emission is the giving off of light energy when a molecule moves from an excited state back to its ground state. Eyepiece  the part of the microscope that the user looks through to view the image. Eyepieces also add extra magnification, usually 10× or 15×. Eyepieces can be high eyepoint or low eyepoint. High-eyepoint eyepieces are for users who wear glasses. Low eyepoint are for users who don’t wear glasses. Excited state  when an electron receives energy and leaves the ground state. Exposure  in imaging, exposure is the length of time that light is allowed onto the sample or onto the CCD. Exposure is also used to describe the perceived level of brightness in the image. Images that are overexposed appear too bright. Images that are too dark are underexposed.

F Field of view (abbrev. FOV)  in microscopy, the field of view is the area that the user can see down the eyepieces, or on the monitor if using a digital system. Eyepieces have a slightly narrower field of view than that of the objective lens. This is to cut out the areas around the very edge of the image where there might be dark edges or vignetting. Filter  an attachment added to the light path that enhances or eliminates a particular wavelength of light. Filters are used to enhance colours and features in the sample. Fine focus knob  the dial used to make small movements of the stage or objective (depending on the model of microscope) in the Z axis for focusing. Fine focus is usually done while looking down the eyepieces. See also Course focus knob. Focal point  the point in space at which the light rays come together to create the image. Focus  the Latin ‘focus’ meaning ‘domestic hearth’. In optics, the focus is the point where all the light rays have converged to a single point. For an image to be ‘in focus’, it must have clear and sharp definition around the edges of the objects in the image. FOV  see Field of view.

G Graticule  see Reticle. Greenough stereomicroscope  a model of stereomicroscope where the light enters two smaller objective lenses, positioned side by side, achieving binocular vision. The light is then directed into the two separate eyepieces. Ground state  the lowest energy level of an atom or electron.

H High-eyepoint eyepieces  High-eyepoint eyepieces are designed for users who wear glasses. The eyepieces project the image further out, so that the focal point is higher than in loweyepoint eyepieces.

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I Immersion oil  when imaging with an oil objective, immersion oil must be added to funnel the light into the objective lens. The immersion oil must have the same refractive index as the objective lens. Incandescent bulb  a bulb that emits light by running an electrical current through a resistant material such as tungsten. The resistance creates kinetic energy, which produces light and heat. Inverted microscope  a model of microscope that has the light source and condenser above the stage and the objective below the stage. The light shines downwards through the sample and into the objective. Iris  see Diaphragm.

J JPEG (Joint Photographic Experts Group)  JPEG is a lossy compression format. It is a good medium between image quality and file size. JPEG is suitable for images that are going to be used in a presentation or as a reference image.

K Koehler illumination  a method of illuminating the sample in an even and consistent way, across the field of view.

L Lambda (λ)  the 11th letter of the Greek alphabet. In optics it is used to denote wavelength. LED  see Light-emitting diode. Lens  the Latin ‘lens’ meaning ‘lentil’, as the lens has a similar shape to a lentil. In optics, a lens is a curved piece of glass or crystal that bends light and magnifies or focuses the light rays. Lenses are defined by their curvature. See Concave, Convex, Biconcave, and Biconvex. In biology, the lens of the eye is similarly responsible for bending and focusing light, so that it lands focused on the retina. This is achieved by the muscles in the eye tensing and relaxing to change the curvature of the lens. Light  from the Latin ‘lux’ meaning ‘light’, or the Greek ‘leukos’, meaning ‘white’. Light is a form of electromagnetic energy that is made of photons. Light in the visible spectrum is used for light microscopy; there are other imaging techniques that use light outside the visible spectrum. Some techniques, such as Electron Microscopy, use electrons instead of light. Light-emitting diode  a semiconductor that, when a current is passed through it, provides a non-heating source of light. LEDs are an increasingly common light source for microscopy.

160 Glossary Light year  in astronomy, a light year is the distance light travels in 1 year. One light year equals 9.4607 × 1012 km. Lossless compression  a compression algorithm used on digital images to reduce the file size without losing any of the original information. The quality of the image is maintained. Lossy compression  a compression algorithm used in digital images to reduce the file size. Some of the original information is lost. If a high level of lossy compression is applied to an image, it will result in loss of image quality. Low-eyepoint eyepieces  Low-eyepoint eyepieces are designed for users who do not wear glasses. The focal point is lower than in high-eyepoint eyepieces and the users eyes are positioned closer to the eyepiece during viewing.

M Magnification  from the Latin ‘magnus’ meaning ‘great’ and ‘facere’ meaning ‘to make or to do’. Magnification is the perceived increase of the size of an image or object. The job of the microscope is to magnify the sample to an extent where the user can see the samples form and features. Magnification is achieved by bending the rays of light using the lenses of the objective and eyepieces. In microscopy, the magnification is denoted by ‘×’. For example, ten times magnification is 10×. Media  (see Medium) Medium (pl. media)  in physics, medium is a substance that something is carried in. For example, air is a medium, water is a medium. If light moves from air into water, it can be said that the light moved from one medium into another medium. Micrometre (pronounced my-crom-i-ter)  a glass slide with a precision scale bar etched or printed onto it. Used for calibration and scale measurement. Micrometre (pronounced my-cro-mee-ter) (abbrev. μm)  from the Greek ‘mikros’ meaning ‘small’ and ‘metron’ meaning ‘measure’. One micrometre equals one millionth of a metre. Microtome  a lab machine used for cutting thin slices of tissue. Monocular head  from the Latin ‘monoculus’ meaning ‘having one eye’. A monocular head on a microscope has one eyepiece, as opposed to a binocular head which has two.

N NA  see Numerical aperture. Nanometre (abbrev. nm)  from the Greek ‘nanos’ meaning ‘dwarf’ and ‘metron’ meaning ‘measure’. One nanometre equals one thousand-millionth of a metre. Wavelength is measured in nm. Numerical aperture (abbrev. NA)  the range of angles from which a lens can receive light.

O Objective lens  from the Latin ‘objectivus’ meaning ‘object’. The objective lens is the part of the microscope that provides the majority of the magnification for the image. An objective contains two or more lenses.

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Objective head  the part of the microscope that the objective lenses are attached to. The objective head can hold several objective lenses of different magnifications. The user selects the objective lens of choice by rotating the objective head. Optic nerve  the nerve that connects the eye to the brain. Optical zoom  magnification that is achieved using the lenses of the microscope. Optical zoom is a true form of magnification. Image quality is not lost as the zoom increases. See also Digital zoom. Overexposure  an image is considered overexposed when the light levels are too bright. In image capture, overexposure is a result of the sample being exposed to too much light, or to light for too long. Overexposure washes out the image and results in loss of detail and colour.

P Parfocal  Parfocality is when the image stays in focus when the magnification changes. In microscopy, this means that the image stays in focus when the user swaps between objectives. Petrographic microscope  From the Greek ‘petra’ meaning ‘rock’ or ‘petros’ meaning ‘stone’. Petrographic microscopy is a technique used by scientists to examine the composition of rock samples. Phase annulus  a solid disc with a transparent ring in it. One of the inserts needed for Phase Contrast microscopy. Phase Contrast  a light microscopy technique where ambient light is used to illuminate the sample. The light in phase is very gentle, making it a suitable technique for imaging live samples. The light approaching from the sides provides contrast to otherwise transparent samples, meaning that stains do not need to be used. Phase plate  the phase plate is one of the filters required for phase imaging. The phase plate consists of a clear circular lens with a dark circle marked on it. It works in conjunction with the phase annular. Photon  from the Greek ‘phōs’ or ‘phōt’ meaning ‘light’. A photon is an individual particle of light. Photons can act individually or move in a group as a wave. Photoreceptor  from the Greek ‘phōs’ or ‘phōt’ meaning ‘light’ and the Latin ‘recipere’ meaning ‘receive’. The photoreceptor is the mechanical device that detects photons. An example of a photoreceptor is a CCD. The rod and cone cells in the eye are also photoreceptors. Photosensitive cells  from the Greek ‘phōs’ or ‘phōt’ meaning ‘light’ and the Latin ‘sentire’ meaning ‘to feel’. In biology, photosensitive cells detect photons and convert the photon signal into an electrical signal. Examples of photosensitive cells are the rod and cone cells in the eye. Planar  from the Latin ‘plānāris’ meaning ‘flat’. In microscopy, planar is used to describe an objective lens that has been corrected to reduce vignetting by increasing the flatter area in the centre of the lens. Plano-concave lens  from the Latin ‘plānāris’ meaning ‘flat’ and ‘cavus’ meaning ‘hollow’. A plano-concave lens is a lens that curves inwards on one side and is flat on the other side. Plano-convex lens  from the Latin ‘plānāris’ meaning ‘flat’, and ‘convexus’ meaning ‘vaulted’ or ‘arched’. A plano-convex lens is a lens that curves outwards on one side and is flat on the other. Prism  from the Greek ‘prisma’ meaning ‘thing sawn’. A prism is a geometric piece of glass or crystal that is used to bend light. In microscopy, prisms are often used to direct light into the eyepieces or to send light in a different direction.

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R Reflected light  light that bounces off the sample and into the objective. For example, darkfield is a technique that uses reflected light.  Reflection  from the Latin ‘reflectere’ meaning ‘to bend back’. In optics, reflection is defined as light being sent in a different direction when encountering a change in medium. Refraction  from the Latin ‘refringere’ meaning ‘break up’. In optics, refraction is the slowing down and bending of light. The extent to which a substance can bend light is known as the refractive index. Materials with a high refractive index bend the light a large amount. Materials with a low refractive index bend the light a small amount. The lenses in the objective and eyepieces refract the light to create magnification and focus. Refractive index  a measurement of the extent to which light is bent when moving from one medium to another. Resolution  from the Latin ‘resolvere’ meaning ‘loosen or release’. In microscopy, resolution is the scale at which two small objects can be discerned as separate objects. At low resolution it can be difficult to tell small or close together objects apart. At high resolution, the seperation is clear.  Reticle  from the Latin ‘reticulum’, meaning ‘net’. The reticule is also known as a graticule. A glass insert in the eyepiece that can be used for scale and measurement when looking at the sample. Retina  from the Latin ‘rete’ meaning ‘net’. The retina is the area found in the back of the eye that contains the photosensitive cells. Rods  a photosensitive cell in the eye that is highly sensitive to light. They can detect lower light levels and are responsible for night vision.

S Scatter (photon scatter)  when light moves from a medium of lower density to a medium of higher density (e.g. moves from air into water), the photons encounter a sudden resistance and are scattered. This dissipates the light and reduces brightness. Depending on how the photons are scattered, this can also change the colour of the light. For example, light travelling from space suddenly encounters the atmosphere. Blue light is easily scattered by the atmosphere, thereby making the sky appear blue. Sine wave  from the Latin ‘sinus’ meaning ‘wave’. A sine wave is a measurable and repeating curve that shows consistent oscillation occurring over time. Spherical aberration  from the Greek ‘sphaira’ meaning ‘ball’. A spherical aberration is caused by the misalignment of lenses, resulting in the light rays not converging at the focal point. Spherical aberrations can be recognised as an inability to focus the image. Spontaneous emission  spontaneous emission is when an atom or electron moves from an excited state, back to the ground state. As this occurs, photons are emitted. Stage  the platform where the sample sits. Some microscopes focus and navigate the sample by moving the stage, others by moving the objective. Movement of the stage in the Z axis is done using the focus knobs (see Course and Fine focus). Movement of the stage in the X and Y axis is done using the stage controls. Stereomicroscope (also known as dissecting microscope)  a type of low to medium magnification microscope that can be used for working with larger samples and performing tasks such as dissections, manufacturing, and soldering.

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Stereopsis  from the Greek ‘stereo’ meaning solid and ‘opsis’ meaning ‘sight’. Stereopsis is the ability to perceive depth which comes from having binocular vision. Stereo vision  see Binocular vision. Stimulated emission  in optics, stimulated emission is when a molecule is being excited by one wavelength of light and emitting light of a lesser wavelength.

T Tetrachromat/tetrachromy/tetrachrome  from the Greek ‘tettares’ meaning ‘four’ and ‘khrōma’ meaning ‘colour’. To be tetrachromat means to have four different types of cone cell. Tetrachromes can see light outside of the human visual range, usually into the ultraviolet range. There is a genetic condition that affects a very tiny percentage of women, which makes them tetrachromatic. TIFF (Tagged Image File Format)  TIFF files are a commonly used format. Compression is lossless, meaning the file will be large but will maintain maximum quality. Images for publication will often look best if TIFF is used, and some data analysis will require TIFF images as they carry large amounts of information. Transmitted light  light that passes through the sample and into the objective. Brightfield uses transmitted light. Trichromat/trichromy/trichrome  from the Greek ‘treis’ meaning ‘three’ and ‘khrōma’ meaning ‘colour’. To be trichromat means to have three different types of cone cell. Humans are trichromes as we have red, blue, and green cone cells. Trinocular head  from the Greek ‘treis’ meaning ‘three’ and ‘oculus’ meaning ‘eye’. A trinocular head on a microscope has three paths for the light to travel. Two go to the eyepieces and one goes to the camera or CCD. Using a trinocular head allows the user to add a camera to the system.

U Underexposure  underexposure is when the image has not received enough light. In image capture, an image is underexposed when the sample did not receive strong enough light or was not exposed to the light for long enough. This results in loss of colour and fine detail in the image.

V Vignetting  an optical aberration where the edges of the field of view appear darker and out of focus. Vignetting is usually caused by the lenses in the system being misaligned, although all lenses will a have certain level of vignetting around the edges where refraction is strongest.

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W Wave  photons of light travel and move together as a wave. Wavelength  in a sine wave, the wavelength is the distance between two peaks (technically known as the spatial period). The wavelength of light is measured in nanometres and is denoted by the symbol lambda, λ. WD  see Working distance. White light  all the colours of light combined make white light. Any of the individual colours of light can be extracted from white light. Working distance (abbrev. WD)  the distance the objective needs to be from the sample in order to focus. The shorter the working distance, the higher the numerical aperture. As magnification increases, working distance decreases.

X X axis  the X axis is the axis going from left to right when sitting in front of the microscope. It is generally the long axis of the slide, although this can be different on different systems.

Y Y axis  the Y axis is the axis going from back to front (near and far) when sitting in front of the microscope. It is generally the short axis of the slide, although this can be different on different systems.

Z Z axis  the Z axis is the axis that the stage moves up and down in. Z-stacking  the imaging technique where several images of the sample are captured while moving through the Z axis. Z-stacked images can then be composited to make a false 3D image of the sample. Zoom  see Digital zoom and Optical zoom.