Biophysical Methods Tools and Techniques in Biology

Biophysical Methods Tools and Techniques in Biology Part I-Microscopy

Dr. Nisha Raghava Dr. Ravindra Pratap Raghava Associate Professor, FIST-DST department of Botany, SMM Town PG College, Ballia (UP), India.

Associate Professor & Head, FIST-DST department of Botany, SMM Town PG College, Ballia (UP), India.

First Published by Scholarink 2017

Old No. 38, New No. 6 McNichols Road, Chetpet Chennai - 600 031 An imprint of Notion Press Media Private Limited

Copyright © Nisha Raghava 2017 © Ravindra Pratap Raghava 2017 ISBN 978-1-948147-49-1

This book has been published with all reasonable efforts taken to make the material error-free after the consent of the author. No part of this book shall be used, reproduced in any manner whatsoever without written permission from the author, except in the case of brief quotations embodied in critical articles and reviews. The Author of this book is solely responsible and liable for its content including but not limited to the views, representations, descriptions, statements, information, opinions and references [“Content”]. The Content of this book shall not constitute or be construed or deemed to reflect the opinion or expression of the Publisher or Editor. Neither the Publisher nor Editor endorse or approve the Content of this book or guarantee the reliability, accuracy or completeness of the Content published herein and do not make any representations or warranties of any kind, express or implied, including but not limited to the implied warranties of merchantability, fitness for a particular purpose. The Publisher and Editor shall not be liable whatsoever for any errors, omissions, whether such errors or omissions result from negligence, accident, or any other cause or claims for loss or damages of any kind, including without limitation, indirect or consequential loss or damage arising out of use, inability to use, or about the reliability, accuracy or sufficiency of the information contained in this book.

Dedicated To Our Parents And Teachers

Contents

List of Figures and Tables ix Forewordxi Prefacexiii 1. Introduction to Microscopy 1.1 History of the Microscope 2. Optical Microscopy Techniques (Light Microscopy) 2.1 Principle 2.2 The Optical Lens 2.3 Microscope Vocabulary 2.4 Types of Light Microscopy 3. Electron Microscopy 3.1 Little history: electrons? 3.2 Instrument 3.3 Types of Electron Microscopy 3.4 Reactions Exploited In SEM 3.5 Reactions Exploited In TEM 3.6 SEM-TEM 4. X-Ray and Scanning Probe Microscopy 4.1 X-Ray Microscopy (Figure 32) 4.2 Confocal Laser Scanning Microscopy (CLSM) 5. Safety and Handling of Microscopes 5.1 Safety with the Compound Microscope 5.2 How to use the microscopes properly? 5.3 Maintenance  5.4 Safety with the Electron Microscope

1 1 5 5 6 8 9 55 55 55 56 59 62 63 71 71 73 83 83 83 84 85

Appendix: Microscopy Terminology 87 Suggested Readings 99 Important Microscope Companies in India and World 101 Biophysical Methods: Tools and Techniques in Biology Upcoming-Topics Covered in Parts 103 Bibliography105 109 Author Index Subject Index 111

List of Figures and Tables

Figures Figure 1 Historical figures of Microscopes from 16th to 18th century 3 Figure 2a A convex lens is thickest in the middle. Light that is travelling parallel 6 to the optical axis of the lens. Figure 2b A concave lens is thinnest at the middle. Light that is travelling parallel to the optical axis of the lens will diverge after passing the lens. The light will then appear to come from the focal point in front of the lens. 7 Figure 3a An object placed at a distance S1 in front of a convex lens produces a real image of the object if S1 > f.7 If S1 < f then the image cannot be projected on a screen and is called Figure 3b  a virtual image. This image will, for an observer to the right of the lens, look like it came from the position S2 in front of the lens. This is the 8 principle behind a magnifying glass. Figure 4a Beck Dissecting Microscope 10 Figure 4b The ‘Queckett’ Dissecting Microscope 10 11 Figure 4c The Binocular Wooden Dissecting Microscope Figure 4d Watson & Sons Dissecting Microscope 11 12 Figure 4e Carl Zeiss Horseshoe Dissecting Microscope Figure 4f James Swift & sons Compound Dissecting Microscope 13 Figure 4g Simple Dissecting Microscopes 13 14 Figure 4h Simple Dissecting Microscopes with detachable hands Figure 5a Differences between a Simple Microscope and a Compound Microscope 15 16 Figure 5b Schematic presentation of a simple optical microscope Major components of a light microscope 19 Figure 6 Figure 7 Light Microscopy- Compound Microscope 19 20 Figure 8a Showing working distance Figure 8b Working of Immersion oil lens 21 Figure 9a Left-ZEISS Stemi 508; Right-ZEISS Stemi 508 22 Figure 9b Components of Stereoscopic Microscope 23 Figure 10 Components and Ray diagram of Dark-Field Microscope 25 Figure 11 Components and Ray diagram of Phase contrast microscope 28 Figure 12 Schematic Diagram of a DIC microscope 31 Figure 13 Interaction of polarized light with an anisotropic crystal 33 Figure 14 Hypothetical field of view in plane polarized light showing the shape of the indicatrix section for five uniaxial positive crystals. 33 Figure 15 Components and configuration of Polarized Light Microscope 34 Figure 16 Cut-away diagram of an upright microscope equipped both for transmitted light and epi-fluorescence microscopy. 36 Figure 17 Microscope filter cube containing the exciter and barrier filters as well as the dichromatic mirror. 38

x

List of Figures and Tables

Figure 18 The principles behind epi-fluorescence microscopy. Excitation and emission filter as well as the dichroic mirror is in practice placed 40 in one unit called the filter cube. Where obj. means the objective Figure 19 The principle and design of total internal reflection fluorescence 43 (TIRF) microscope  Figure 20a A UV microscope actually contains two entirely different optical systems, one for UV and the other for visible light, built into the same microscope 47 48 Figure 20b Schematic diagram of the reflected light microscope system.  Figure 21 Schematic of a conventional IR microscope operating in transmission mode and employing Schwarzschild reflecting optics and confocal apertures 51 51 Figure 22 An Infared Microspectrometer Figure 23 Concept of a near-field microscope with a photoinduced transient aperture. 52 53 Figure 24 Schematic presentation of the experimental setup for IR microscope Figure 25 Illustration of the electron gun 58 59 Figure 26 Effects produced by electron bombardment of a material Figure 27 The Scanning Electron Microscope 64 Figure 28 Geometry of SEM 64 65 Figure 29 Schematic presentation of Image formation in SEM 67 Figure 30 The Transmission Electron Microscope Figure 31 Transmission electron microscope with all of its components 68 Figure 32 Soft X-Ray Microscopy  72 Figure 33 Schematic diagram of the working mechanism of confocal 77 laser scanning microscopy Figure 34 Jablonski diagram 79

Photoplates Photoplate 1 Histological sections of plant tissues with birefringent objects Photoplate 2 SEM secondary electron image. Pollen - various types Photoplate 3 SEM backscattered electron image

35 60 61

Tables Table 1 Table 2 Table 3

Visibility of different objects through unaided eyes and microscopes Difference between simple disecting and a compound microscope Fluorescence of Naturally Occurring Substances

4 15 45

Foreword

I am privileged to write a foreword note for this book which was a challenge of condensing, organizing, and searching all the available information and knowledge in the field specially in the days of explosion of progress and advancement of technology. In recent years Physical methods for qualitative and quantitative analysis along with the structural elaborations have become the integral part of all branches of Sciences. This is especially true for biological sciences, where these are incorporated as Biophysical Methods. There is a paradox in the growth of scientific knowledge, in case any information accumulates in ever more intimidating quantity, disconnected facts and impenetrable mysteries give way to rational explanations, and gradually the essential principles of a subject come into focus. Among the illustrations of modern techniques in biological sciences, a tremendous progress has been noticed in the field of microscopy by the invention of more sophisticated microscopes. Presently, in the era of modern applied sciences and techniques, a vacuum was noticed for a book on Microscopy. This book under the title “Biophysical Methods: Tools and Techniques in Biology,” attempts to furnish basic aspects of Microscopy in Part 1, in a condensed and organized manner. The progress in the field of biological techniques must have made the task of authors more demanding in projecting information that best represents in this field. They have clearly ensured that. The strength of the book lies primarily with the outstanding research and teaching experience of the authors who have contributed their expertise and historical perspectives to this complex effort along with latest available information on the subject. They deserve all the credit for selecting the relevant stuff that best represents the true conceptual advances in the field of Microscopy. All the topics are adequately covered and presented in a uniform style and level of clearity. The text in general is divisible into various sections as historical background, types of microscopy with detailed account technically including figures, tables and diagrams. Title related all the topics are adequately discussed. The book also deals with technical terms used in microscopy, separately in the form of Appendix. This part of the book is intended to provide the students of biological sciences with a concise general account of the microscopy. This book is principally for students and researchers in the area including the medical students. It is written in a way that even a stranger to this field could allow in by starting at the beginning. On the other hand, I hope that it will also be useful to working scientists in search of a guide to help them pick their way through a vast field of knowledge.

xiiForeword

Finally, the love and support for each other among the authors sustained them during the whole venture whenever the road became bumpy. Dr. Nisha Raghava and Dr. R. P. Raghava are to be congratulated for the excellent job they have done in writing this book on Microscopy. I hope the book will be beneficial for the students of under graduation, post graduation, research and also students preparing for medical and various competitions. I also wish them good luck for their upcoming parts of this series in advance. October 13th 2017

Yogesh Kumar Sharma Professor and Former Head Officer Incharge-All India Coordinated ICAR Research project on Micronutrients and Secondary Pollutant Elements Program Coordinator-Centre of Excellence Depatment of Botany Lucknow University

Preface

Throughout the writing of this book our goal has been how to teach the beginner how to use microscopes. In thinking about a cover, our initial plan was to suggest the title “Practical Use of Microscopes.” However, the needs of the scientific community for a more comprehensive reference and the furious pace of electronic imaging technologies demanded something more. Practitioners of microscopy have long required an instructional text to help align and use a microscope— one that also reviews basic principles of the different optical modes and gives instructions on how to match filters and fluorescent dyes, choose a camera, and acquire and print a microscope image. Advances in science and technology have also profoundly changed the face of light microscopy over the past ten years. Instead of microscope and film camera, the light microscope is now commonly integrated with a projectors, CCD camera, computer, software, and printer into electronic imaging systems. Therefore, to use a modern research microscope, it is clear that research scientists need to know not only how to align the microscope optics, but also how to acquire electronic images and perform image processing. Thus, the focus of the book is on the integrated microscope system, with foundations in optical theory but extensions into electronic imaging. Accordingly, the title has been chosen “Biophysical Methods: Techniques and Tools in Biology, Part I- Microscopy.” The book covers three areas: Historical backgrounds, optical principles involved in diffraction and image formation in the light microscope; the basic modes of light microscopy and electron microscopy; the components of modern electronic imaging systems and the basic image-processing operations that are required to prepare an image. Each chapter is introduced with theory regarding the topic at hand, followed by descriptions of instrument alignment and image interpretation. As biologist and practitioner of microscopy, the reader will notice that we have focused on how to align and operate microscopes and have given somewhat abbreviated treatment to the physical theory and principles involved. The content is not difficult, so the demonstrations are valuable aids in absorbing essential optical principles. They also allow time to pause and reflect on the economy and aesthetic beauty of optical laws and principles. If carried out, the demonstrations and exercises also offer opportunities to become acquainted with new biological specimens that the reader may not have confronted or seen before by a new mode of light microscopy. A basic glossary has also been included to aid readers not already familiar with complex terminology. Finally, because the text

xivPreface

contains several detailed descriptions of theory and equipment that could be considered ancillary, an effort has been made to subordinate these sections so as to not obscure the major message. Special thanks are due to many individuals who made this work possible. Foremost we thank profoundly our son, Kanu Yash Pratap, who encouraged us in this work and devoted much time to reading the text and providing much assistance in organizing content, selecting figures, and editing text. We also thank the many students who have taken microscope courses over the years, who inspired us to write the book and gave valuable advice. In particular, we would like to thank Prof. D. Banerji, Ex. Head of the Botany Department at CCS University, Meerut, who suggested us through every phrase and equation to get the facts straight and to clarify the order of presentation. We would also like to thank and acknowledge the help of many colleagues who provided helpful criticisms and corrections to drafts of the text. We express our gratitude to Prof. Yogesh Kumar Sharma, for kindly consenting to write the Foreword. Finally, we thank scholarink.com (Publishers and Distributors) as well as the Printers for their great patience in receiving the manuscript and managing the production of the book. Nisha Raghava & Ravindra P Raghava, Associate Professors, FIST-DST PG department of Botany, SMM Town PG College, Ballia-277001, Uttar Pradesh, India.

Introduction to Microscopy

1

The meaning of Microscope is “little seer” or “seer of little things.” Cells are small and in almost all situations a microscope is needed to observe them and their subcellular components. Microscope as the name suggests are instruments that help to enlarge minute (micro = very small) organisms or their parts. A microscope not only enlarges or magnifies the object but also ‘resolves’ it, that is makes it possible to differentiate between two points present close together in the objects being viewed. These minute cells cannot be seen through our naked eyes because our eyes have a limited resolving power and the objects smaller than 100 µ (0.1 mm) cannot be distinguished. The earliest type of simple microscope used to examine small objects was in the form of glass globes filled with water. Through the microscopes, which are either provided with simple or combination of lenses, the resolving power is increased. The minute objects get several times magnified and is easily seen. In fact the invention of the microscope led to the discovery and description of cells by Hooke in 1655. The microscope is still an extremely important tool in biological research. The light microscope has a limited capability in regards to the size of a particle that can be examined. The electron microscope provides additional resolution that allows for the examination of sub-cellular structures and even molecules.

1.1

History of the Microscope

The history of microscope can be traced from 8th century B.C., when in Ninevels (an ancient city in Assyria) a rock crystal was excavated and was used as “lens.” In 5th Century B.C., Greek Philosopher, Euclid investigated the properties of curved reflecting surface which were used later on in the microscope. A Roman philosopher, Lucius Seneca (4 B.C.–65 A.D.) stated that water filled glass globules would assist in seeing small objects. A Greco-Egyptian astronomer, Claudius Ptolemy (127–151 A.D.) studied some problems of magnification by using curved surfaces. The use of convex lenses for magnification of small objects

Biophysical Methods Tools and Techniques in Biology

The book covers three areas: Historical background, optical principles involved in diffraction and image formation in the light microscope; the basic modes of light microscopy and electron microscopy; the components of modern electronic imaging systems and the basic image-processing operations that are required to prepare an image. Each chapter is introduced with theory regarding the topic at hand, followed by descriptions of instrument alignment and image interpretation. As a biologist and a practitioner of microscopy, the reader will observe that the book is focused on how to align and operate microscopes with an abbreviated treatment to the physical theory and principles involved. The content is not difficult, so the demonstrations are valuable aids in absorbing essential optical principles. They also allow time to pause and reflect on the economy and aesthetic beauty of optical laws and principles. If carried out, the demonstrations and exercises also offer opportunities to help the reader become acquainted with new biological specimens using a new mode of light microscopy. A basic glossary has also been included to aid readers not already familiar with complex terminology. DR. NISHA RAGHAVA (M.Sc., M.Phil., PhD., FBS, FISPP) Associate Professor, FIST-DST sponsored, PG Department of Botany, S.M.M. Town (PG) College, Ballia - 277001, U.P. (Affiliated to Jannayak Chandrashekhar University, Ballia, Uttar Pradesh). She obtained her M.Sc and M.Phil degrees from IAS, Meerut University, Meerut and her Ph.D degree under the guidance of Dr. Pramod Kumar from Rohilkhand University, Bareilly. She has more than three decades of research and teaching experience. Her list of publications includes 35 research papers and 25 review articles in National and International Journals. She has been a lifelong member of several Botanical Societies of India and has guided four research students through their Ph.D. DR. RAVINDRA PRATAP RAGHAVA (M.Sc., M.Phil., PhD., FBS, FISPP, MNASc) Associate Professor & Head, FIST-DST sponsored, PG Department of Botany, S.M.M.Town (PG) College, Ballia - 277001, U.P. (Affiliated to Jannayak Chandrashekhar University, Ballia, Uttar Pradesh). He obtained his M.Sc, M.Phil & Ph.D degrees under the guidance of Prof. Y. S. Murty (FNA), from IAS, Meerut University, Meerut. He has more than three decades of research and teaching experience. His list of publications includes 50 research papers and 25 review articles in National and International Journals. He has been a lifelong member of many Botanical Societies of India and has guided nine research students through their Ph.D. Price 200