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Table of contents :
Front Cover
Inside Front Cover
Stevens & Lowe's Human Histology
Copyright Page
Table Of Contents
About This Book
Summary Headings
Figure Legends
Assessment
Student Consult Website
Feedback
Preface
Acknowledgements
Dedication
1 Histology
Introduction
Cells Are Basic Functional Units
Techniques Used in Histology and Cell Biology
Light Microscopy
Tissue Staining
Electron Microscopy
Confocal Microscopy
Virtual Microscopy Uses Digitalized Light Microscopic Slides
2 The Cell
Introduction
Cell Membranes
Transport in and Out of Cells
Cytosol
The Nucleus
Mitochondria
Endoplasmic Reticulum (ER) and Golgi
Vesicles
Cytoskeleton
Cell Inclusions and Storage Products
Cell Division
Cell Death
3 Epithelial Cells
Introduction
Epithelial Cell Junctions
Epithelial Cell Surface Specializations
Secretory Adaptations
Barrier Function of Epithelium
4 Support Cells and the Extracellular Matrix
Introduction
Extracellular Matrix
Basement Membrane and External Lamina
Cell Adhesion to Extracellular Matrix
Support Cell Family
5 Contractile Cells
Introduction
Skeletal Muscle
Cardiac Muscle
Smooth Muscle
Myofibroblasts
Pericytes
Myoepithelial Cells
6 Nervous Tissue
Introduction
Nerve Cells (Neurons)
Myelin
Central Nervous System
Peripheral Nervous System
7 Blood Cells
Introduction
Bone Marrow–Derived Stem Cells
Methods of Studying the Blood Cells
Red Blood Cells
White Blood Cells
Platelets
Haematopoiesis
Bone Marrow
8 Immune System
Introduction
Lymphocytes
Macrophages and Dendritic Cells
Bone Marrow
Thymus
Lymph Nodes
Spleen
Mucosa-Associated Lymphoid Tissue
9 Blood and Lymphatic Circulatory Systems and Heart
Introduction
Blood Circulatory System
Systemic Blood Vessels
Portal Blood Systems
Lymphatic Circulatory System
Stem Cells and the Vasculature
The Heart
Stem Cells and the Heart
10 Respiratory System
Introduction
Upper Respiratory Tract
Larynx
Distal Respiratory Tract
Pulmonary Vasculature
Pleura
11 Alimentary Tract
Introduction
Oral Cavity and Its Contents
Teeth
Introduction
Dentinogenesis and Odontoblasts
Ameloblasts and Enamel Formation
Cementum and Periodontal Ligament
Tooth Development
The Gums
Salivary Glands
Transport Passages
Oesophagus
Introduction
Anal Canal
Digestive Tract
Stomach
Small Intestine
Introduction
Exocrine Pancreas
Large Intestine
Appendix
12 Liver
Introduction
Liver Vasculature
Hepatocytes
Hepatocyte Stem Cells and Liver Regeneration
Functional Organization of Hepatocytes
Intrahepatic Biliary Tree
Gallbladder
Introduction
13 Musculoskeletal System
Introduction
Skeletal Muscle
Muscle Attachments
Bone
Introduction
Bone Cells
Mineralization of Osteoid
Bone Remodelling
Joints
Introduction
14 Endocrine System
Introduction
Endocrine Cell and Tissue Specialization
Pituitary
Anterior Pituitary
Posterior Pituitary
Hypothalamus
Pineal Gland
Thyroid Gland
Introduction
Parathyroid
Adrenals
Introduction
Adrenal Cortex
Adrenal Medulla
Pancreas
Introduction
Ovary and Testis
Diffuse Neuroendocrine System
Introduction
Paraganglia
15 Urinary System
Introduction
Outline of the Urinary System
Kidney Structure
Kidney Function
Kidney Vasculature
Renal Microcirculation
Nephron
Glomerulus
Glomerular Filtration Barrier
Mesangium
Tubular and Collecting System
Introduction
Juxtaglomerular Apparatus
Erythropoietin Synthesis
Lymphatic Drainage and Nerve Supply of the Kidney
Lower Urinary Tract
16 Male Reproductive System
Introduction
Testes
Anatomy and Development
Seminiferous Tubules
Sertoli Cells
Epididymis
Vas Deferens
Seminal Vesicles
Prostate
Introduction
Bulbourethral Glands
Penis
Endocrine Control
17 Female Reproductive System
Introduction
Mons Pubis, Labia Majora and Labia Minora
Clitoris
Vagina
Uterus
Cervix
The Epithelial Content of the Cervix
Uterine Body
Uterine Tubes
Ovary
Introduction
Gamete Production and Maturation in the Ovary
Menstrual Cycle
Introduction
Pregnancy
Trophoblast
Introduction
Decidua
18 Skin and Breast
Introduction
Epidermis
Non-keratinizing Epidermal Cells
Skin Appendages
Pilosebaceous Apparatus
Eccrine Sweat Glands and Ducts
Apocrine Glands
Dermis
Subcutaneous Tissue
Features of Skin in Different Sites
Breast
Nipple
Breast Structure: Lobes and Lobules
Breast Development
Breast Changes in Pregnancy
19 Special Senses
Introduction
Ear
Eye
Accessory Components of the Eye
Case Studies
Chapter 2
Case 2.1 A Child With Muscle Weakness
Case 2.1 Answer
Case 2.2 A Tumour of Unknown Origin
Case 2.2 Answer
Chapter 3
Case 3.1 Nodules on the Liver
Case 3.1 Answer
Case 3.2 A Girl With a Blistering Rash
Case 3.2 Answer
Chapter 4
Case 4.1 Infant With Broken Femur
Case 4.1 Answer
Type I
Type II
Type III
Type IV
Chapter 5
Case 5.1 Sudden Cardiac Death
Case 5.1 Answer
Chapter 6
Case 6.1 Possible Epileptic Seizure
Case 6.1 Answer
Case 6.2 Diabetes-Related Neuropathy
Case 6.2 Answer
Chapter 7
Case 7.1 A Man Who Was Tired and Weak
Case 7.1 Answer
Chapter 8
Case 8.1 A Boy With Big Lymph Nodes
Case 8.1 Answer
Chapter 9
Case 9.1 Sudden Death in an Obese Woman
Case 9.1 Answer
Case 9.2 A Man With Central Chest Pain
Case 9.2 Answer
Chapter 10
Case 10.1 A Case of Anosmia
Case 10.1 Answer
Case 10.2 A Case of Hoarse Voice
Case 10.2 Answer
Case 10.3 A Case of Deteriorating Chest Infection
Case 10.3 Answer
Chapter 11
Case 11.1 An Obese Man With Heartburn
Case 11.1 Answer
Case 11.2 A Young Man With Persistent Severe Diarrhoea
Case 11.2 Answer
Case 11.3 A Woman With Abdominal Discomfort, Diarrhoea and Weight Loss
Case 11.3 Answer
Chapter 12
Case 12.1 A Man With a Bad Liver
Case 12.1 Answer
Chapter 13
Case 13.1 A Case of a Road Traffic Accident
Case 13.1 Answer
Case 13.2 Muscle Weakness in Childhood
Case 13.2 Answer
Case 13.3 A Man With a Limp
Case 13.3 Answer
Case 13.4 A Woman With Back, Buttock and Leg Pain
Case 13.4 Answer
Chapter 14
Case 14.1 A Woman With Multiple Symptoms
Case 14.1 Answer
Case 14.2 A Man Who Became Obese and Hypertensive
Case 14.2 Answer
Chapter 15
Case 15.1 A Child With Blood in the Urine
Case 15.1 Answer
Case 15.2 A Middle-Aged Man With Severe Proteinuria
Case 15.2 Answer
Case 15.3 A Young Man With Multiple Injuries
Case 15.3 Answer
Chapter 16
Case 16.1 A Man With Urinary Problems
Case 16.1 Answer
Case 16.2 A Couple With Subfertility
Case 16.2 Answer
Chapter 17
Case 17.1 A Woman Who Cannot Become Pregnant
Case 17.1 Answer
Chapter 18
Case 18.1 A Man With Blisters
Case 18.1 Answer
Case 18.2 A Woman With a Lump in the Breast
Case 18.2 Answer
Chapter 19
Case 19.1 Gradual Onset of Deafness
Case 19.1 Answer
Case 19.2 A Woman With Deteriorating Vision
Case 19.2 Answer
Review Questions
Chapter 1 Histology
Chapter 2 The Cell
Chapter 3 Epithelial Cells
Chapter 4 Support Cells and the Extracellular Matrix
Chapter 5 Contractile Cells
Chapter 6 Nervous Tissue
Chapter 7 Blood Cells
Chapter 8 Immune System
Chapter 9 Blood and Lymphatic Circulatory Systems and Heart
Chapter 10 Respiratory System
Chapter 11 Alimentary Tract
Chapter 12 Liver
Chapter 13 Musculoskeletal System
Chapter 14 Endocrine System
Chapter 15 Urinary System
Chapter 16 Male Reproductive System
Chapter 17 Female Reproductive System
Chapter 18 Skin and Breast
Chapter 19 Special Senses
Review Answers
Chapter 1 Histology
Chapter 2 The Cell
Chapter 3 Epithelial Cells
Chapter 4 Support Cells and the Extracellular Matrix
Chapter 5 Contractile Cells
Chapter 6 Nervous Tissue
Chapter 7 Blood Cells
Chapter 8 Immune System
Chapter 9 Blood and Lymphatic Circulatory Systems and Heart
Chapter 10 Respiratory System
Chapter 11 Alimentary Tract
Chapter 12 Liver
Chapter 13 Musculoskeletal System
Chapter 14 Endocrine System
Chapter 15 Urinary System
Chapter 16 Male Reproductive System
Chapter 17 Female Reproductive System
Chapter 18 Skin and Breast
Chapter 19 Special Senses
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
R
S
T
U
V
W
Z
Inside Back Cover
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Stevens & Lowe’s
HUMAN HISTOLOGY
Cover images: Diagram of proximal convoluted tubule from kidney. Histology of a cancer derived from epithelial cells. Histology of juxtaglomerular apparatus from kidney. Histology of epididymal duct containing spermatozoa.
FIFTH EDITION
Stevens & Lowe’s
HUMAN HISTOLOGY James S. Lowe, BMedSci, BMBS, DM, FRCPath Emeritus Professor of Neuropathology School of Medicine University of Nottingham Nottingham, UK
Peter G. Anderson, DVM, PhD
Professor, Department of Pathology University of Alabama at Birmingham, School of Medicine Birmingham, AL, USA
Susan I. Anderson, BSc, MMedSci, PhD Professor of Pathology Deputy Head and Director of Teaching and Learning Division of Medical Sciences & Graduate Entry Medicine School of Medicine Faculty of Medicine & Health Sciences University of Nottingham Royal Derby Hospital Centre Nottingham, UK For additional online content visit StudentConsult.com
© 2020, Elsevier Limited. All rights reserved. First edition © 1992, Gower Medical Publishing Second edition © 1997, Times Mirror International Publishers Third edition © 2005 by Mosby, an imprint of Elsevier Limited. Fourth edition © 2015 by Mosby, an imprint of Elsevier Limited. The right of James Lowe, Peter Anderson, and Susan Anderson to be identified as authors of this work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).
Notices Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Although all advertising material is expected to conform to ethical (medical) standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or the value of such product or the claims made of it by its manufacturer. ISBN: 978-0-323-61279-1
Editorial Assistant: Susana Sainz García Content Strategist: Alexandra Mortimer Content Development Specialist: Trinity Hutton Project Manager: Umarani Natarajan Design: Patrick Ferguson Illustration Manager: Teresa McBryan Marketing Manager: Michele Milano
Printed in China Last digit is the print number: 9 8 7 6 5 4 3 2 1
CONTENTS About This Book, vi Preface, vii Acknowledgements, viii Dedication, ix 1 Histology, 1 2 The Cell, 12 3 Epithelial Cells, 41 4 Support Cells and the Extracellular Matrix, 56 5 Contractile Cells, 71
11 Alimentary Tract, 177 12 Liver, 209 13 Musculoskeletal System, 222 14 Endocrine System, 246 15 Urinary System, 267 16 Male Reproductive System, 297 17 Female Reproductive System, 314
6 Nervous Tissue, 83
18 Skin and Breast, 338
7 Blood Cells, 103
19 Special Senses, 358
8 Immune System, 120
Case Studies, 376 Review Questions, 394 Review Answers, 411 Index, 414
9 Blood and Lymphatic Circulatory Systems and Heart, 140 10 Respiratory System, 158
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ABOUT THIS BOOK Considerable thought has gone into designing this book to meet the requirements of students who have limited time in which to assimilate information, yet need to take in maximum detail without re-reading portions of text or becoming fatigued. Many features of this book have been designed to ease reading and assimilation and to highlight clinical relevance, which we hope will facilitate understanding and thus make it easier to remember details. There is a saying that goes: ‘Memorization is what we do when what we are trying to learn makes no sense’ (Anonymous). Our goal with this revised textbook is to make it easier for students to understand histology in a relevant context so that they do not have to resort to rote memorization.
Summary Headings These headings (bold-faced declarative sentences in boxes scattered throughout) provide a summary of the forthcoming text, giving a quick overview of the whole section. These have proven to be popular with students as a high-yield overview of each section.
Figure Legends The figure legends are, in general, not repetitive of the main text and are designed to be read when referenced from the main text. This serves two purposes: first, it maintains the flow of information and, second, it provides a refreshing break from reading the main text. For this reason, many of the captions are used as a vehicle to explain complex pieces of information, particularly those relating to three-dimensional structure.
CLINICAL EXAMPLE BOXES We have chosen many clinical examples to illustrate the vital role that an understanding of histological structure will play in subsequent studies of human biology, disease and clinical practice.
PRACTICAL HISTOLOGY BOXES Many students give up microscopy because they feel that they cannot see what they have just read about. The practical histology sections are designed to put histology into a classroom teaching perspective; we hope that this will lessen the anxiety of the students who feel that they cannot use a microscope.
KEY FACTS BOXES These boxes provide a number of the most important Key Facts relating to the subject just covered. They are ideal for pre-examination panic states to get a student’s thoughts in-line and focused on the most important concepts. Throughout the book you will see that small sections of text have been emboldened. These are problem-based ‘hooks’ – text that will be of particular use to readers using the book as a reference for problem-based or case-based study.
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ADVANCED CONCEPT BOXES In these sections we supply more advanced knowledge than is strictly necessary for an understanding of the basic principles. These sections often contain the results of up-to-date research, including some of the most important elements of progress in cell biology.
ASSESSMENT We have put in a section of brief self assessment questions towards the back of the book. These should help track your understanding. We have also put a series of case studies and answers online, together with expanded self review questions, at the Student Consult website supporting the textbook. Please visit https:// studentconsult.inkling.com.
STUDENT CONSULT WEBSITE We understand that many students benefit from having online access to text and images from their textbooks so that they can be looked at when convenient. The Student Consult site provides this as well as other great learning resources, such as the Virtual Histolab. You will find more images to explore plus video tutorials on practical histology from each of the chapters. If you have not already registered with the site, do it now.
FEEDBACK Many of the changes in the presentation of this book have been stimulated by comments about previous editions made by teachers and students who used the book for their courses and personal study. This has proved so successful that we again wish to canvas the comments of the users of the fifth edition in the hopes of further improving it in subsequent editions. We hope that teachers and students will again take this opportunity to have an input in the creation of this valuable teaching resource and are happy to receive suggestions about any new material and illustrations that could be included. We can be contacted by letter at the address given but are also happy to receive comments by e-mail at: [email protected].
P R E FA C E We are delighted to present this fifth edition of Human Histology which sees Professor Susan Anderson on the authorship team. Susan brings a breadth of knowledge in microscopy and the teaching of histology, pathology and ultrastructure, as well as being the Honorary Secretary for Education and Outreach of the Royal Microscopical Society. In writing this preface, we have looked back at the original thinking that drove creating the first edition over 25 years ago. Our thoughts are as follows: • Studying histology equips students with vocabulary, knowledge and insights that improve their learning and understanding of cell biology, anatomy, physiology, pathology and medicine. • Students benefit from using a textbook which is user-friendly, nicely organised, and well-illustrated (despite wide availability of learning resources on the web). • There is a lot that could go into a textbook and it is easy to overburden the reader. When faced with the conflict between being comprehensive and being comprehensible, we have chosen comprehensible. • We have stuck with our principles of using only human material and aligning the text to that which happens in humans. The book is not aimed at those wanting to know about rodents, fish or flies. Things continue to change as biological discoveries are made and new concepts emerge. Wider use of confocal microscopy, computer-aided microscopy and imaging in living material has added significantly to knowledge about the structure and function of cells and tissues. This revision, as with the previous editions, has had a main aim of updating the text and images accordingly.
The place of histology in curricula is also changing. We have reviewed the text to match the changes in teaching in medicine, dentistry, biomedical sciences, nursing and midwifery courses. We have included more links to clinical contexts where knowledge of histology helps clinical understanding. It is our experience that the students who demonstrate a good grasp of histology stand out in their wider, contextualized understanding of their subject. We hope that the excellence of layout linked to its readability and ‘high yield’ presentation will keep the book a favourite for both students and teachers. The learning resources are online, which we hope will help teachers of the subject present human material of direct relevance to their classes. Self-assessment and review items are online, which we hope will help students in their studies. We have been delighted to note the emergence of integrated first degree and Masters level courses that have a major component of histology. We have tried to ensure that this book is well suited to such courses by maintaining a systematic coverage of the subject. The fact that computer-based imaging and high throughput systems for histology are being routinely applied in biomedical research implies a need for people with knowledge of what is being viewed. A core competence in histology is increasingly in demand. We have enjoyed revising the book and we hope you enjoy using it. Among the many updates and changes we have put in is some of the exciting ‘new stuff ’ which we predict will become mainstream in understanding about health and diseases in the future. Watch this space…
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AC K N OW L E D G E M E N T S Once again, we take this opportunity to give our thanks to the many colleagues who made contributions to the earlier editions of the book. In the preparation of this fifth edition we want to thank the following people for their generous help: Dr. Ian Todd works in cellular immunopathology at the University of Nottingham where he has expertise in autoimmune diseases and the fascinating group of autoinflammatory diseases. We thank Ian for his generosity in commenting on the chapter related to the immune system. His expertise as a professional immunologist and teacher has been invaluable. Dr. Jan Kosinski and Dr. Martin Beck work at the Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. This unit is doing amazing things in resolving the structure of complex biological systems. The fabulous images of the nuclear pore complex were generously provided by them from their work. Dr. Laura Antón-Sánchez of the Departamento de Inteligencia Artificial, Universidad Politécnica de Madrid, is one of the ‘next generation’ people applying artificial intelligence to computer analysis of images in histology. Laura kindly helped with the magnificent 3D images of nerve cells in the chapter on the nervous system. Professor Wolfgang Jakob Streit of the Department of Neuroscience, University of Florida, works on the role of microglial cells during brain aging and in the development of Alzheimer’s disease. Jake very kindly provided the images of human microglial cells in the chapter on the nervous system. Dr. Kate Keller works at the Casey Eye Institute, Oregon Health & Science University, Portland. Kate has been studying the novel structure of tunneling nanotubes in her work on the eye disease glaucoma. We are very grateful to Kate for providing the images of one cell sending things into another cell along a tunnel for Chapter 2. Professor Vishnu V. B. Reddy, MD, Hematopathologist from the Department of Pathology at the University of Alabama, Birmingham generously provided expert content suggestions and images for Chapter 7 (Blood Cells). We gratefully acknowledge the Human Protein Atlas (www.proteinatlas.org) for the ability to use material under a version of the creative commons license. (See Uhlén M et al, 2015. Tissuebased map of the human proteome. Science PubMed: 25613900 DOI: 10.1126/science.1260419.) Several images in the book have been reproduced from original publications, cited in the relevant captions. We thank the authors and their publishers for permission to reproduce these images using the RightsLink service of the Copyright Clearance Centre (http://www.copyright. com). We would especially like to extend our sincere thanks to the team from Elsevier, especially Alexandra Mortimer, Trinity Hutton and Umarani Natarajan, who greatly assisted in our work and were very patient when things got a bit slow. Finally, the contribution of Dr. Alan Stevens to this book cannot be overstated. Over the years Steve has contributed to many textbooks on histology, pathology, dermatopathology and histotechnology. Although not on the authorship team for this edition, he still takes a keen interest. To answer a question he asked last month: yes, it is still great fun. We are extremely grateful to Steve for his contributions as teacher, author, friend and wine consultant.
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It is easy to become completely immersed in interesting projects afforded to us in our work. What saves us are the family, friends and others who tolerate our mental absences, support us, and make our lives complete. To these we dedicate this book, with love and gratitude. To Peter and Ita Anderson for unwavering belief and support for their daughter, Susan. To our significant others: Joan Anderson, Susana Sainz García, and Brendan Devaney To our children: Nicholas and William Lowe, Robert and Lindsey Anderson, Matthew Anderson, and Katherine and Emily Devaney And to the owners and staff in several establishments in Cambados, Galicia who provided sustenance to one of the authors (JL) during the revision of this book by serving him fine Albariño, wonderful local seafood and the occasional vermút rojo when he was most in need of refreshment. JL PA SA
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1 Histology INTRODUCTION Histology is the study of the microscopic structure of biological material and the ways in which individual components are structurally and functionally related. Histology is central to biological and medical sciences, standing at the crossroads between a range of disciplines.
• Anatomy: sometimes referred to as microscopic anatomy or microanatomy, histology places gross anatomy into context, enabling the understanding of cell and tissue microstructure and how it relates to function. • Physiological and life sciences: physiology, pharmacology, cell biology and biochemistry, among others; histology links them together providing a point of reference that is between gross anatomy and these disciplines and explains the structure underpinning function and treatment. • Pathology: knowledge of histology makes it easier to predict, understand and treat the functional deficits that are brought about by structural changes resulting from physiological adaptations or disease. Knowledge of normal histological appearance is essential if abnormal diseased structures are to be recognized, and to comprehend how abnormal biochemical and physiological processes result in disease. Samples of human biological material can be obtained from many areas of the body by quick, safe biopsy techniques (Fig. 1.1), using instruments such as: • Scalpels for directly accessible tissues such as the skin, mouth and nose • Needles into solid organs • Endoscopic tubes into the alimentary tract or body cavities • Special flexible cannulae inside blood vessels. In modern medicine, despite sophisticated imaging and genetic testing, a histological diagnosis is still the mainstay or the ‘gold standard’ of clinical practice. This is illustrated via two case studies in Fig. 1.2. Histology is a dynamic and evolving science
Modern investigative techniques have revolutionized our understanding of cells and tissues. The techniques of electron and confocal microscopy, cloning of cells in culture, protein sequencing and molecular genetics have also given unprecedented insight into the working of cells. This research has been translated into practice, enabling significant advances in diagnostic pathology
Needle biopsy brain eye thyroid lymph nodes breast lung and pleura liver kidney bone and bone marrow testis skeletal muscle
Endoscopic biopsy respiratory tract trachea bronchus lung alimentary tract oesophagus stomach small intestine colon and rectum urinary tract mainly bladder
Also transvascular biopsy heart, liver direct excision biopsy skin, mouth, larynx, uterine cervix curettage biopsy endometrial lining of uterus
Fig. 1.1 Histology in Diagnostic Medicine. Small samples are obtained from many areas of the body by various techniques. Histological examination of such biopsies is an increasingly important and direct way of diagnosing disease.
and in developing new and targeted treatment. An example is tissue microarray (TMA) research, in which markers in tiny cores of tissue from tumours of many patients are investigated. These studies are giving new insights into tumour biology. Personalized medicine recognizes that even similar tumours are unique: two breast tumours can have different expression of oncogenes and markedly different receptor expression (see Fig. 18.23). Knowing more about the characteristics of a particular tumour enables targeted treatment of individuals in a bespoke manner, appropriate to their tumour biology, rather than treating all tumours of an organ as one disease. 1
2
CHAPTER 1 Histology
CLINICAL EXAMPLE Histology in Disease Diagnosis Case Study 1 A 20-year-old student develops kidney failure and the cause is not apparent from blood tests or radiology findings. The renal physician therefore removes a piece of kidney via a needle biopsy so that the diagnosis can be made by histological examination, electron microscopy and immunofluorescence. Special staining methods highlight subtle structural abnormalities by light microscopy (see Fig. 1.2), Electron microscopy provides valuable information about abnormalities at a subcellular level and fluorescence microscopy allows the detection of immune complex deposition contributing to the pathogenesis of the disease. Based on the abnormalities that are revealed, an accurate histological diagnosis is determined and the renal physician can institute appropriate treatment. Clinical management of this patient’s condition requires knowledge of the microanatomy of the kidney. Assessment of progress and the effectiveness of treatment are monitored by repeat biopsy. Case study 2 A 15-year-old girl has swollen lymph nodes in her neck. A surgeon removes one so that it can be examined histologically, and microscopy reveals that the swelling is caused by a form of cancer. The classification of tumours is determined by histology and immunohistochemistry. Accurate histological assessment of tumours is the cornerstone of modern cancer treatment, since the treatment given to this girl depends on the histological type of the tumour (i.e. whether it is derived from muscle, lymphoid cells, or endocrine cells). Thus, the pathologist’s report, which depends on the histological assessment of that specific tumour being accurate, will determine the type of chemotherapy deemed to be most efficacious for each cancer patient’s treatment protocol.
a
b Fig. 1.2 Kidney. (a) Percutaneous needle biopsy sections from a diagnostic case. (b) High-power view of these needle biopsy specimens of kidney (paraffin section, martius scarlet blue trichrome [MSB] stain). The special stain shows the nature and location of the main abnormality, destruction of the afferent arteriole of the glomerulus by a disease process called ‘fibrinoid necrosis’ (arrow).
Some histological terminology persists from a time before cell and tissue function was understood.
The first microscopic investigations of biological material were carried out by Robert Hooke in his seminal publication Micrographia, just over 300 years ago. The study of histology began with the development of simple light microscopes and techniques for preparing thin slices of biological material to make them suitable for examination. Such studies led Virchow to propound his cellular theory of the structure of living organisms that established the cell as the basic building block of most biological material. Each cell was considered as an individual unit surrounded by a wall called the cell membrane and containing within it all the machinery for its function. Early microscopists had limited understanding of physiology and function and named features by their appearance or eponymously; for example, the little canals linking osteocytes in bone are called canaliculi and the macrophagelike cells in the liver Küppfer cells. This can be frustrating as there is a lot of vocabulary to learn and the system does not easily accommodate new information; however, it is slowly evolving.
CELLS ARE BASIC FUNCTIONAL UNITS The cell is the basic unit of structure of most living organisms and cells vary considerably. Although all cells in the human body are ultimately derived from a single fertilized ovum, each cell develops structural attributes to suit its function through the process of differentiation. Molecular biology has shown that cells of diverse morphological appearance can be grouped together because of common functional attributes or interactions. The general structural and biological properties of cells are discussed in Chapter 2. Some cells are adaptable. It has also become apparent that even in adults, there are populations of highly adaptable, uncommitted stem cells, which can modify both their structure and their functional activity to adapt to changing environmental demands. This facility is of vital importance in adaptation to internal or external stress and is seen in normal and disease processes, for example, enlargement (hypertrophy) of a skeletal muscle cell in response to exercise, a reduction in size (atrophy) due to inactivity, or increasing numbers of cells (hyperplasia) in a gland due to excess stimulation. Some cells are constantly being replaced by this population of stem cells and are able to repopulate tissues after injury – for example, the outer layers of skin. Other cells cannot regenerate in a meaningful way; loss of such cells leads to the formation of a replacement tissue that fills the defect but is non-functional (e.g. replacement of damaged heart muscle by strong fibrous tissue following a heart attack).
CHAPTER 1 Histology
Cell group
Epithelial cells
Support cells
Contractile cells
Nerve cells
Germ cells
Blood cells
Immune cells
Hormonesecreting cells
Example
gut and blood vessel lining, covering skin
fibrous support tissue, cartilage, bone
muscle
brain
spermatozoa
circulating red and white blood cells
lymphoid tissues and white cells (nodes and spleen)
thyroid and adrenal
Function
barrier, absorption, secretion
organize and maintain body structure
movement
direct cell communication
reproduction
oxygen transport, defence
defence
indirect cell communication
Special features
tightly bound together by cell junctions (see Chapter 3)
produce and interact with extracellular matrix material (see Chapter 4)
filamentous proteins cause contraction (see Chapter 5)
release chemical messengers on to surface of other cells (see Chapter 6)
half normal chromosome complement (see Chapters 16 and 17)
proteins bind oxygen, proteins destroy bacteria (see Chapter 7)
recognize and destroy foreign material (see Chapter 8)
secrete chemical messengers (see Chapter 14)
3
Fig. 1.3 Functional Cell Classification.
Cells are classified according to their main function
The following groupings are used in this book: epithelial cells, support cells, contractile cells, nerve cells, germ cells, blood cells, immune cells and hormone-secreting cells (Fig. 1.3). It is important, however, to recognize that a cell may have several functions and be a member of more than one cell group. For example: • Many of the hormone-producing cells are also epithelial in type, being tightly bound together by specialized junctions to form a gland • Many immune cells are also blood cells • Some support cells are also contractile. The structural and functional specializations delineating each type of cell group are broadly outlined in Chapters 3–8 and are discussed in more detail throughout the book. Tissues are functional arrangements of cells
Tissues are discrete, organized collections of cells having similar morphological characteristics. These were traditionally subdivided into four types: • Epithelial tissues, or cells that cover surfaces, line body cavities, or form solid glands such as salivary glands • Muscular tissues, or cells with contractile properties • Nervous tissues referred to cells forming the brain, spinal cord and nerves • Connective tissue, a term used widely to describe a wide range of living material characterized by a dominant extracellular matrix component and the associated cells that produce the matrix. In theory, its function is to act as a supporting stroma, serving more highly specialized cell types. The original group of ‘connective tissues’ included cell/matrix combinations, such as bone, cartilage, tendon, fibrous tissue,
adipose tissue, bone marrow and blood. It has also been traditional to use the term ‘loose areolar connective tissue’ to describe tissue that is partly made up of support cells that produce an extracellular matrix, but which also contains cells belonging to the immune system (e.g. lymphocytes and macrophages), nerve cells and blood vessels. In this book, the term ‘connective tissue’ has been avoided because it underemphasizes the structural organization involved in this group of highly developed tissues. Instead, the concept of support cells is used, which emphasizes the importance of interactions between extracellular matrix and cells. Support cells and their specializations are discussed in Chapter 4, while bone, cartilage, tendons and ligaments are discussed in Chapter 13. In some cases, the cells forming a tissue are all of the same structure, forming simple tissues; for example, fat cells forming adipose tissue. However, most apparently distinct tissues contain a mixture of cells with different functions, which may be termed compound tissues (Fig. 1.4). For example, ‘nervous tissue’ contains nerve cells (neurons), support cells (astrocytes), immune cells (microglia) and epithelial cells (ependyma). The concept of simple and compound tissues is useful in descriptive histology, but for brevity, the unqualified term ‘tissue’ is used to imply either type. Tissues form organs and systems
An organ – for example the heart, liver, or kidney – is an anatomically distinct group of tissues, usually of several types, which perform specific functions. For example, in the organ skin, the tissue types involved are an outer epithelium – the epidermis – overlying the dermis made of supporting connective tissue, and containing the mechanical, immunological and nutrient support, and nerve endings, and housing the skin appendages. Deep to this is the subcutis, which is composed primarily of
4
CHAPTER 1 Histology
TECHNIQUES USED IN HISTOLOGY AND CELL BIOLOGY cells
simple tissues
compound tissue (e.g. cortex of kidney)
organ (e.g. kidney)
system (e.g. urinary system)
Fig. 1.4 Cells, Tissues, Organs and Systems.
adipose tissue. In the intestines of the gastrointestinal (GI) tract, these organs are composed of layers of tissues from the lumen outward including a lining epithelium, layers of underlying support tissue followed by external smooth muscle arranged to facilitate peristalsis. Peristalsis (the coordinated muscle contractions propelling food along the intestine) and glandular secretions are controlled by a range of nerve plexi. The term ‘system’ can be used to: • Describe cells with a similar function but widely distributed in several anatomic sites • Describe a group of organs that have similar or related functional roles. The specialized hormone-producing cells scattered in the gut and lung (diffuse endocrine system) cannot be an organ, as they do not form an anatomically distinct mass, whereas the tongue, oesophagus, stomach, intestines, exocrine pancreas and rectum are components of the ‘alimentary system’, and the kidney, pelvicalyceal system, ureters and bladder are part of the ‘urinary system’. The relationships between cells, tissues, organs and systems are shown in Fig. 1.4.
The most common way to study cells is by light microscopy. Tissues are mounted on glass slides as thin preparations, stained with appropriate dyes, illuminated by light and viewed using glass lenses. This is called histology, or histopathology, if biopsy material from diseased tissue is being investigated. The analysis of the fine structure of cells, for example, blood cells or those obtained via cervical smear, by light microscopy is referred to as cytology. There is a limit to the detail that can be resolved using light microscopy, with very small structures within cells being invisible. Electron microscopy uses a beam of electrons instead of light, which greatly increases resolution, allowing the subcellular composition of cells to be defined. These techniques are complemented by the increasing use of immunohistochemical methods. Antibodies are applied to specific cell constituents to visualize details within cells at the light microscopic level that are not visible by other techniques. For example, the location of specific proteins or subcellular components can now be defined in light microscopic preparations by using immunohistochemical staining techniques. Complementary to this, immunofluorescence techniques enable molecules to be visualized using antibodies linked to fluorescent probes. Use of a fluorescence microscope with the appropriate filters to produce a wavelength of light that excites the fluorescently tagged antibody facilitates viewing of a range of dyes, and the advent of confocal microscopy has greatly enhanced the resolution possible with light microscopy (Fig. 1.6) It is also possible to demonstrate specific DNA and RNA sequences by the technique of in situ hybridization, thereby gaining fundamental insight into the molecular working of cells.
Light Microscopy Light microscopy using wax-embedded sections is the main technique used in histology
Routine light microscopy uses thin sections of tissue to study cell morphology. Resolution of structures by light microscopy is of the order of 0.2 µm, but in routine practice with paraffin sections resolution seldom exceeds 0.6 µm. Sections are usually obtained from paraffin wax-impregnated tissues (see box on p. 5 for detail) using a microtome. Sections are mounted on a glass microscope slide for staining. In routine laboratory practice, it generally takes 24 hours to produce a wax section for histology. In some cases (e.g. surgical biopsies), it is necessary to look at fresh tissues that have not been exposed to protein crosslinking in fixation. In this situation, tissue is made sufficiently firm to cut by freezing, a technique referred to as preparing a frozen section (see box on p. 5 for detail).
CHAPTER 1 Histology
Tissue Staining To see tissue detail, it is necessary to stain the tissue components in a histological section
Cells are virtually colourless and thus sections need to be stained for light microscopy. To enable staining, the wax is removed from the sections with an organic solvent before the section is rehydrated through increasing dilutions of alcohol in water. When fully rehydrated, the sections are stained with any of a number of stains, some of which are listed below. There are four main types of staining: • Empirical • Histochemical • Enzyme histochemical • Immunohistochemical. Empirical stains are widely used and form the basis of most routine stains in histology and histopathology
Many of the stains have been discovered by trial and error over a period of 100 years or more, and many methods use dyes and principles (e.g. use of mordants to fix a dye in a material) that were developed by the textile industry. In most cases, the precise
ADVANCED CONCEPT
5
details of the mechanism of the specific linkage between dye and tissue is not fully understood: sometimes it appears to be related to the sizes of the dye molecules used and sometimes the result of ionic charges on the dye molecules. The most common empirical stain is haematoxylin and eosin (H&E), a simple, reliable and inexpensive method of producing sections with blue nuclei and pink/red cytoplasm. Most of the micrographs in this book are stained with H&E. Other empirical stains include van Gieson’s and trichrome methods (see ‘Advanced Concept’ box on p. 6). In some cases, staining is the result of a specific chemical reaction between a specific tissue component and a component of the stain solution; these methods are called histochemical methods. In histochemical methods, specific chemical compounds within the tissue can be localized
A commonly used example of a simple histochemical staining method is the PAS (or periodic acid–schiff) reaction, which demonstrates a wide range of tissue carbohydrates, including cytoplasmic glycogen and complex carbohydrate-containing substances, such as epithelial mucins. The rationale of the method is that carbon-to-carbon bonds in 1,2-glycols are cleaved using an oxidative agent, periodic acid. This produces dialdehydes, which then react with the colourless Schiff’s reagent (fuchsin–sulphurous acid) to produce a vivid magenta-coloured compound.
ADVANCED CONCEPT
Frozen Sections
Paraffin Embedding
The process of fixation and embedding of biological material in paraffin and other media may destroy certain components, particularly enzymes and some antigenic sites. If frozen water is used as the supporting medium, these components are better preserved and can be demonstrated by suitable techniques. Fresh (unfixed) material is rapidly frozen to −150°C to −170°C by immersion in, for example, liquid nitrogen, so that it hardens to a solid mass owing to freezing of tissue water. Thin sections (5–10 µm) are then cut on a special microtome housed in a refrigerated cabinet (a cryostat) and stained without exposure to alcohol or other organic solvents. Frozen sections are used to demonstrate the cellular localization of enzymes and soluble lipids, and in the identification of substances using immunofluorescence and immunocytochemical methods. There are obvious advantages to preparing frozen sections including speed and retention of antigenicity, although without appropriate consideration, ice crystal damage can damage the tissue, thereby reducing resolution. Further use is made of frozen sections in diagnostic histopathology, when an urgent tissue diagnosis is required of, for example a suspected tumour, while the patient is still on the operating table. In skilled hands, a frozen section of a sample of human tissue stained with H&E can be prepared and examined under the microscope within 5 min of its removal from the body. In this way, a rapid and accurate histological diagnosis can be established while the patient is in the operating theatre, enabling the appropriate surgical room procedure to be performed.
Paraffin embedding is the standard method of preparing thin sections of biological material for histological examination by light microscopy. It is cheap, comparatively simple and lends itself to automation. The sample is preserved by immersion in a fixative, usually in an aqueous formalin-based fixative solution, which cross-links or precipitates proteins and prevents degradation of the tissue. It is then progressively dehydrated by passage through a series of alcohol solutions (e.g. 60%, 70%, 90% and 100%) until all water (intrinsic tissue water and fixative water) has been removed and the specimen is thoroughly permeated with absolute alcohol. The alcohol is then replaced by an organic solvent (e.g. xylene), which is miscible both with alcohol and with molten liquid paraffin wax (alcohol is not miscible with paraffin wax). The resulting specimen is immersed in paraffin wax at a temperature just above the melting point of the wax, which is solid at normal working room temperature. When the biological material is thoroughly permeated by the molten wax, it is allowed to cool so that the wax solidifies. The wax acts as a physical support to the sample, allowing thin sections (2–8 µm) to be cut using a microtome, without deformation of the cellular structure and architecture.
6
CHAPTER 1 Histology
ADVANCED CONCEPT Commonly Used Histological Stains Haematoxylin and Eosin (H&E) The combination of the two dyes, haematoxylin (blue) and eosin (red), is the most useful stain for the examination of biological material; the staining is simple to perform, reliable, inexpensive and informative. Cell nuclei stain blue (depending on section thickness and the formulation of haematoxylin used), and most components of the cell cytoplasm stain pink/red. Van Gieson Method The simple van Gieson method stains collagen pinkish-red and muscle yellow (see Fig. 10.21); it is commonly used in combination with a stain for elastic fibres. The elastic van Gieson (EVG) stain is valuable for demonstrating and differentiating the common support cell fibres, particularly elastic fibres, which stain brownblack, and collagen fibres, which stain pinkish-red; muscle is stained yellow (see Fig. 10.21). Trichrome Methods The trichrome methods employ a mixture of three dyes to stain different components in different colours. There are many trichrome methods, and they can be used to demonstrate general architecture, to emphasize support fibres, or to distinguish support fibres from muscle fibres. An important use of a trichrome method is the demonstration of the cellular, osteoid and mineralized components of bone in non-decalcified bone embedded in acrylic resin (see Figs 13.17a and 13.19b).
Alcian Blue Method The Alcian blue dye method is used mainly to demonstrate acidic mucins secreted by some epithelial cells (see Fig. 11.44b) and can be combined with the PAS reaction to distinguish between acidic and neutral epithelial mucins. Through control of pH or other variables in the staining solution, the Alcian blue method can be used to demonstrate the extracellular glycosaminoglycan matrix (see Fig. 4.14d) of support cells. May–Grünwald–Giemsa Method The use of the May–Grünwald–Giemsa method is confined mainly to the examination of smear preparations of blood and bone marrow cells. Most of the micrographs in Chapter 7 show red and white blood cells stained by this method. Myelin Methods Several staining techniques can be used to demonstrate normal myelin. The dye solochrome cyanin is used frequently to demonstrate myelin in paraffin sections (see Fig. 6.20). Other methods use modified haematoxylin or osmium tetroxide.
Silver Methods Under appropriate conditions, certain biological components, both within cells and in intercellular materials, reduce silver nitrate to form black deposits of metallic silver at the site of chemical reduction. By modifying the conditions of the silver nitrate solution used, these methods can be used to demonstrate a wide range of structures, including reticular fibres (see Fig. 4.5). Periodic Acid–Schiff (PAS) Method The widely used PAS method has many applications, particularly in the demonstration of various carbohydrates, either alone (e.g. glycogen; Fig. 1.5) or combined with other molecules, such as proteins (e.g. glycoproteins), which are stained magenta. It can therefore be used to delineate basement membranes (see Fig. 4.12a) and some neutral mucins secreted by various secretory epithelial cells. The mucous cells of the stomach are strongly PAS-positive.
Enzyme histochemical techniques identify and localize the sites of activity of particular enzymes
As most biological enzyme systems are labile, they may be destroyed by fixation and tissue processing; thus, most enzyme histochemical methods are carried out on frozen sections. To look at the tissue distribution of specific enzymes, sections of fresh tissue prepared on a cryostat are placed in an incubating solution containing the specific substrate for the enzyme or group of enzymes to be demonstrated, together with any necessary cofactors or inhibitors. The enzyme in the tissue reacts with the substrate to form an insoluble primary reaction product. This is then visualized by its reaction with a visualizing agent, which may be included with the incubating medium or applied as a separate second step.
Fig. 1.5 Liver – Paraffin Section: PAS Stain. This high-power photomicrograph shows intense red staining of the liver cell cytoplasm by the PAS stain, demonstrating the large amounts of glycogen present.
This technique can be used to show the localization of a vast number of enzymes, including acid and alkaline phosphatases, dehydrogenases and ATPases, and is used routinely to detect abnormalities in certain diseased tissue, particularly muscle (see Fig. 13.4). Immunocytochemistry uses antibodies to localize specific proteins in tissue sections
Immunocytochemistry is one of the most important innovations in histology. Antibodies to specific cell molecules are used to detect their presence in tissue sections. These are tagged either directly or indirectly with a product enabling visualization of the labelled molecules. For light microscopy this is a coloured product such as 3,3′-diaminobenzidine (DAB); for fluorescence microscopy the tag emits fluorescence when excited by an
CHAPTER 1 Histology
7
TP
RBC
US BC
M
M AA
EA
a
b
EP G
P1 P2 T
EnC
c
e
appropriate wavelength of light. In electron microscopy, electrondense particles of gold are used. Primary antibodies are either polyclonal or monoclonal. Polyclonal antibodies to a substance are obtained by inoculating an animal (commonly rabbits or sheep) with the purified protein and then harvesting serum from
GBM
d
Fig. 1.6 Comparison of microscopic techniques using a kidney glomerulus visualized by different microscopic methods: (a) wax histology, (b) resin histology, (c) scanning and (d) transmission electron microscopy and (e) confocal microscopy. Different methods show different levels of detail. In a the wax section can be seen arterioles EA and AA, Bowmans capsule lined by epithelium BC, the space where urine flows US, and the tubule down which it leaves the glomerulus TP. The resin section b shows more detail with red blood cells RBC seen in glomerular capillaries and details of mesangial cells M not easily seen in the wax section. The scanning EM image c shows the 3D shape of glomeruli G and tubules T in the renal cortex but not much fine detail. A transmission EM d shows very high resolution and additional detail revealing fine structure of podocytes EP which have foot processes P1 P2 lying on a basement membrane GBM with very thin endothelial cells EnC on the other side. The confocal image e has been stained by immunohistochemistry and shows basement membrane components in green with cell nuclei in purple.
which a specific antibody can be extracted. Alternatively, monoclonal antibody may be produced by inoculating a mouse and fusing suitable antibody-producing cells with immortal mouse myeloma cells to continually produce antibodies in tissue culture.
8
CHAPTER 1 Histology
High-resolution light microscopy can be performed with tissue embedded in resin
Resolution of structures by light microscopy using paraffin sections is seldom better than 0.6 µm, the resolution being limited by the thickness of the section, which is rarely thinner than 3 µm. Much better resolution can be obtained by using thinner sections – about 0.5–2 µm – but these cannot be achieved consistently with wax as the embedding medium and using a standard microtome. The use of harder acrylic and epoxy resins as embedding media allows thinner tissue sections to be cut, thereby allowing increased cellular detail to be seen by increasing resolution (see Fig. 15.7b and c). Acrylic resins are a suitable embedding medium for the production of histological sections of non-decalcified bone
Bone, unless severely diseased, is usually too hard to produce thin histological sections using standard paraffin wax as the embedding medium and normal microtome knives. This is because the difference in hardness between the bone and the wax in which it is embedded is too great, so the bone shatters when the microtome knife blade passes through it, rendering the histology uninterpretable. Bone can be examined histologically in this way only if it is first softened by complete removal of the calcium salts by immersing the fixed bone sample in dilute acid until all the calcium has disappeared; sections can then be produced, but the histology is inevitably modified by the acid treatment. Furthermore, any distinction between mineralized bone and unmineralized osteoid is destroyed by the acid decalcification; this can be important in the diagnosis of some important bone diseases. The problem can be overcome by embedding the bone in an acrylic resin embedding medium (e.g. methylmethacrylate) which, when set (polymerized), has a hardness that is the same as calcified bone, and good sections can be obtained without fragmentation or distortion. Examples are shown in many of the photomicrographs in Chapter 13: for example, see Figs 13.19b and 13.22.
ELECTRON MICROSCOPY An electron microscope uses parallel beams of electrons instead of light waves
In light microscopy, the degree of magnification and resolution achievable is limited by the wavelength of light. Parallel beams of electrons have a much shorter wavelength and if used instead of light, much greater magnification can be achieved. This allows resolution of structures as small as 1 nm, thus permitting the study of subcellular morphology. Two main types of electron
ADVANCED CONCEPT Resins and Histological Embedding Media Acrylic Resin Embedding Certain acrylic resins are used in a manner similar to paraffin wax as embedding media. When set, the resins are harder than paraffin wax and offer more support to the tissue than wax. They have two main advantages over paraffin wax for light microscopy: • With the use of a special microtome, much thinner sections (i.e. 1–2 µm thick) can be obtained than with paraffin wax, giving greater resolution with the light microscope and enabling much more detail to be seen. • They cause very little tissue shrinkage and enable good-quality sections of very hard material to be cut and are therefore used in the histological examination of mineralized bone (see Figs 13.17a and 13.19b). Epoxy Resin Embedding Epoxy resins are the hardest supporting media for biological material. With special sectioning machines, sections as thin as 0.5–1 µm can be cut for high-resolution light microscopy and ultrathin sections, typically 70-90 nm can be prepared for transmission electron microscopy. The transmission electron micrographs in this book were prepared from ultrathin epoxy resin sections. These resins are resistant to the damaging effects of the electron beam in the electron microscope, and continue to support the biological material, whereas other embedding media volatilize in the electron beam. Most of the staining methods used with paraffin and acrylic resin sections cannot penetrate epoxy resins. Fortunately, the stain toluidine blue is an exception, and differentially stains biological components in various shades of blue. The greatest cellular detail obtainable by conventional light microscopy is attained with the use of 0.5–1 µm epoxy resin sections stained with toluidine blue (see Fig. 15.7b and c). Toluidine Blue Stain Toluidine blue is used to demonstrate cells and fibres in very thin epoxy resin sections. Toluidine blue is one of the very few dyes that will penetrate the dense epoxy resin to stain the tissue section. It provides considerable cellular detail, staining the various components of the cells and fibres in the shades of blue in a way that represents their relative electron density; hence the resulting blue picture closely resembles a low-power electron micrograph but is blue instead of black. Prior to preparation of ultrathin sections for electron microscopy, a toluidine blue–stained survey section is obtained from the epoxy resin block to enable the microscopist to trim down the appropriate area of tissue for ultrathin sectioning.
microscopy are used in the study of biological material, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To get the best results in both types of electron microscopy, fixation must be as perfect as can be achieved; this means that a different fixative solution (glutaraldehyde) is used. This must act on the tissues as soon as possible after the tissue sample has been obtained, as subcellular structures can be structurally altered as soon as they become anoxic. For TEM, this is achieved by using very small tissue fragments (