Understanding Light Microscopy

Introduces readers to the enlightening world of the modern light microscope

There have been rapid advances in science and technology over the last decade, and the light microscope, together with the information that it gives about the image, has changed too. Yet the fundamental principles of setting up and using a microscope rests upon unchanging physical principles that have been understood for years. This informative, practical, full-colour guide fills the gap between specialised edited texts on detailed research topics, and introductory books, which concentrate on an optical approach to the light microscope. It also provides comprehensive coverage of confocal microscopy, which has revolutionised light microscopy over the last few decades. 

Written to help the reader understand, set up, and use the often very expensive and complex modern research light microscope properly, Understanding Light Microscopy keeps mathematical formulae to a minimum—containing and explaining them within boxes in the text. Chapters provide in-depth coverage of basic microscope optics and design; ergonomics; illumination; diffraction and image formation; reflected-light, polarised-light, and fluorescence microscopy; deconvolution; TIRF microscopy; FRAP & FRET; super-resolution techniques; biological and materials specimen preparation; and more.

• Gives a didactic introduction to the light microscope
• Encourages readers to use advanced fluorescence and confocal microscopes within a research institute or core microscopy facility
• Features full-colour illustrations and workable practical protocols

Understanding Light Microscopy is intended for any scientist who wishes to understand and use a modern light microscope. It is also ideal as supporting material for a formal taught course, or for individual students to learn the key aspects of light microscopy through their own study.

JEREMY SANDERSON is a Bio-Imaging specialist at the Mammalian Genetics Unit, MRC Harwell Institute, in the UK. He is Chartered Scientist and a Fellow of the Royal Microscopical Society and has served both on the Executive Council and the LM Section Committee.

About the Author ix

Acknowledgements xi

Look-Up Guide to Feature Boxes by Theme xii

Glossary and Definitions xiv

Notes xxiv

Introduction xxvii

1 Our Eyes and the Microscope 1

2 Light 29

3 Basic Microscope Optics 55

4 Microscope Anatomy and Design 75

5 Ergonomics 91

6 Optical Aberrations of the Microscope 101

7 The Microscope Objective 127

8 Condensers and Eyepieces 161

9 Illumination in the Microscope 177

10 Diffraction and Image Formation in Microscopy 211

11 Contrast Generation and Enhancement 243

12 Reflected-Light Microscopy 289

13 Polarised-Light Microscopy: Part 1 – Theory 317

14 Polarised-Light Microscopy: Part 2 – Applied 347

15 Fluorescence Microscopy 383

16 Fluorophores and Fluorescent Proteins 405

17 Optical Sectioning and Confocal Microscopy 425

18 Operating the Confocal Microscope 447

19 Light-Sheet Microscopy 483

20 Bleed-Through and Spectral Unmixing 507

21 Deconvolution 523

22 Multi-Photon Microscopy 543

23 Total Internal Reflection Fluorescence Microscopy 561

24 FRAP and FRET 569

25 Colocalisation 587

26 Super-Resolution Fluorescence Microscopy 613

27 Choosing a Microscope Platform and Core Imaging Facilities 637

28 Biological Specimen Preparation 663

29 Materials Specimen Preparation 687

30 Recording the Image: Part 1 – Theory 707

31 Recording the Image: Part 2 – Applied 733

Appendices

1 Buying, and Tendering for, a Light Microscope 769

2 Troubleshooting Poor Image Quality 773

3 The Michel-Lévy Interference Colour Chart 775

4 Cleaning and Maintenance of the Light Microscope 779

5 Selected Suppliers 783

6 Historical Background 787

7 Timeline of Key Events 791

Index 799

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