Fluorescence Microscopy: From Principles to Biological Applications

This is the first title on the topic designed specifically to allow students and researchers with no background in physics to understand novel light microscopy techniques. Each chapter is written by a renowned expert with many contributions by pioneers in the field, but the editors have ensured that all commonly used methods are covered and that each chapter is comprehensible for non-experts and conforms to the same easily legible standard and style. A companion website with additional examples and video material makes this a valuable teaching resource.

Table of Contents:
Preface XIII List of Contributors XVII 1 Introduction to Optics and Photophysics 1 Rainer Heintzmann 1.1 Interference: Light as a Wave 2 1.2 Two Effects of Interference: Diffraction and Refraction 7 1.3 Optical Elements 14 1.3.1 Lenses 14 1.3.2 Metallic Mirror 17 1.3.3 Dielectric Mirror 18 1.3.4 Pinholes 18 1.3.5 Filters 19 1.3.6 Chromatic Reflectors 20 1.4 The Far-Field, Near-Field, and Evanescent Waves 20 1.5 Optical Aberrations 23 1.6 Physical Background of Fluorescence 24 1.7 Photons, Poisson Statistics, and AntiBunching 30 References 31 2 Principles of Light Microscopy 33 Ulrich Kubitscheck 2.1 Introduction 33 2.2 Construction of Light Microscopes 33 2.2.1 Components of Light Microscopes 33 2.2.2 Imaging Path 34 2.2.3 Magnification 36 2.2.4 Angular and Numerical Aperture 38 2.2.5 Field of View 38 2.2.6 Illumination Beam Path 39 2.3 Wave Optics and Resolution 42 2.3.1 Wave Optical Description of the Imaging Process 43 2.3.2 The Airy Function 47 2.3.3 Point Spread Function and Optical Transfer Function 50 2.3.4 Lateral and Axial Resolution 52 2.3.5 Magnification and Resolution 59 2.3.6 Depth of Field and Depth of Focus 60 2.3.7 Over- and Under Sampling 61 2.4 Apertures, Pupils, and Telecentricity 61 2.5 Microscope Objectives 64 2.5.1 Objective Lens Design 64 2.5.2 Light Collection Efficiency and Image Brightness 68 2.5.3 Objective Lens Classes 73 2.5.4 Immersion Media 73 2.5.5 Special Applications 77 2.6 Contrast 78 2.6.1 Dark Field 80 2.6.2 Phase Contrast 81 2.6.3 Interference Contrast 86 2.6.4 Advanced Topic: Differential Interference Contrast 89 2.7 Summary 94 Acknowledgments 94 References 95 3 Fluorescence Microscopy 97 Jurek W. Dobrucki 3.1 Features of Fluorescence Microscopy 98 3.1.1 Image Contrast 98 3.1.2 Specificity of Fluorescence Labeling 101 3.1.3 Sensitivity of Detection 102 3.2 A Fluorescence Microscope 103 3.2.1 Principle of Operation 103 3.2.2 Sources of Exciting Light 107 3.2.3 Optical Filters in a Fluorescence Microscope 110 3.2.4 Electronic Filters 111 3.2.5 Photodetectors for Fluorescence Microscopy 112 3.2.6 CCD-Charge-Coupled Device 113 3.2.7 Intensified CCD (ICCD) 116 3.2.8 Electron-Multiplying Charge-Coupled Device (EMCCD) 117 3.2.9 CMOS 119 3.2.10 Scientific CMOS (sCMOS) 120 3.2.11 Features of CCD and CMOS Cameras 121 3.2.12 Choosing a Digital Camera for Fluorescence Microscopy 121 3.2.13 Photomultiplier Tube (PMT) 121 3.2.14 Avalanche Photodiode (APD) 122 3.3 Types of Noise in a Digital Microscopy Image 123 3.4 Quantitative Fluorescence Microscopy 127 3.4.1 Measurements of Fluorescence Intensity and Concentration of the Labeled Target 127 3.4.2 Ratiometric Measurements (Ca++, pH) 130 3.4.3 Measurements of Dimensions in 3D Fluorescence Microscopy 131 3.4.4 Measurements of Exciting Light Intensity 132 3.4.5 Technical Tips for Quantitative Fluorescence Microscopy 132 3.5 Limitations of Fluorescence Microscopy 133 3.5.1 Photobleaching 133 3.5.2 Reversible Photobleaching under Oxidizing or Reducing Conditions 134 3.5.3 Phototoxicity 135 3.5.4 Optical Resolution 135 3.5.5 Misrepresentation of Small Objects 137 3.6 Current Avenues of Development 139 References 139 Further Reading 141 Recommended Internet Resources 142 Fluorescent Spectra Database 142 4 Fluorescence Labeling 143 Gerd Ulrich Nienhaus and Karin Nienhaus 4.1 Introduction 143 4.2 Principles of Fluorescence 143 4.3 Key Properties of Fluorescent Labels 144 4.4 Synthetic Fluorophores 149 4.4.1 Organic Dyes 149 4.4.2 Fluorescent Nanoparticles 150 4.4.3 Conjugation Strategies for Synthetic Fluorophores 152 4.4.4 Nonnatural Amino Acids 155 4.4.5 Bringing the Fluorophore to Its Target 156 4.5 Genetically Encoded Labels 158 4.5.1 Phycobiliproteins 158 4.5.2 GFP-Like Proteins 159 4.6 Label Selection for Particular Applications 163 4.6.1 FRET to Monitor Intramolecular Conformational Dynamics 163 4.6.2 Protein Expression in Cells 167 4.6.3 Fluorophores as Sensors inside the Cell 167 4.6.4 Live-Cell Dynamics 168 4.7 Conclusions 168 References 169 5 Confocal Microscopy 175 Nikolaus Naredi-Rainer, Jens Prescher, Achim Hartschuh, and Don C. Lamb 5.1 Introduction 175 5.1.1 Evolution and Limits of Conventional Widefield Microscopy 175 5.1.2 History and Development of Confocal Microscopy 177 5.2 The Theory of Confocal Microscopy 180 5.2.1 The Principle of Confocal Microscopy 180 5.2.2 Radial and Axial Resolution and the Impact of the Pinhole Size 182 5.2.3 Scanning Confocal Imaging 189 5.2.4 Confocal Deconvolution 194 5.3 Applications of Confocal Microscopy 196 5.3.1 Nonscanning Applications 196 5.3.2 Advanced Correlation Techniques 200 5.3.3 Scanning Applications Beyond Imaging 205 Acknowledgments 210 References 210 6 Fluorescence Photobleaching and Photoactivation Techniques 215 Reiner Peters 6.1 Introduction 215 6.2 Basic Concepts and Procedures 216 6.2.1 Putting Photobleaching to Work 216 6.2.2 Setting Up an Instrument 219 6.2.3 Approaching Complexity from Bottom Up 220 6.3 Fluorescence Recovery after Photobleaching (FRAP) 221 6.3.1 Evaluation of Diffusion Measurements 222 6.3.2 Binding 225 6.3.3 Membrane Transport 226 6.4 Continuous Fluorescence Microphotolysis (CFM) 228 6.4.1 Theoretical Background and Data Evaluation 229 6.4.2 Combination of CFM with Other Techniques 231 6.4.3 CFM Variants 232 6.5 Confocal Photobleaching 233 6.5.1 Use of Laser Scanning Microscopes (LSMs) in Photobleaching Experiments 233 6.5.2 New Possibilities Provided by Confocal Photobleaching 234 6.5.3 Artifacts and Remedies 237 6.6 Fluorescence Photoactivation and Dissipation 238 6.6.1 Basic Aspects 238 6.6.2 Theory and Instrumentation 239 6.6.3 Reversible Flux Measurements 239 6.7 Summary and Outlook 241 References 241 7 Forster Resonance Energy Transfer and Fluorescence Lifetime Imaging 245 Fred S. Wouters 7.1 General Introduction 245 7.2 FRET 246 7.2.1 Historical Development of FRET 246 7.2.2 Requirements 254 7.2.3 FRET as a Molecular Ruler 258 7.2.4 Special FRET Conditions 262 7.3 Measuring FRET 265 7.3.1 Spectral Changes 266 7.3.2 Decay Kinetics 272 7.4 FLIM 280 7.4.1 Frequency-Domain FLIM 282 7.4.2 Time-Domain FLIM 283 7.5 Analysis and Pitfalls 285 7.5.1 Average Lifetime, Multiple Lifetime Fitting 285 7.5.2 From FRET/Lifetime to Species 286 Summary 287 References 288 8 Single-Molecule Microscopy in the Life Sciences 293 Markus Axmann, Josef Madl, and Gerhard J. Schutz 8.1 Encircling the Problem 293 8.2 What Is the Unique Information? 295 8.2.1 Kinetics Can Be Directly Resolved 295 8.2.2 Full Probability Distributions Can Be Measured 296 8.2.3 Structures Can Be Related to Functional States 297 8.2.4 Structures Can Be Imaged at Superresolution 298 8.2.5 Bioanalysis Can Be Extended Down to the Single-Molecule Level 300 8.3 Building a Single-Molecule Microscope 301 8.3.1 Microscopes/Objectives 301 8.3.2 Light Source 304 8.3.3 Detector 310 8.4 Analyzing Single-Molecule Signals: Position, Orientation, Color, and Brightness 316 8.4.1 Localizing in Two Dimensions 316 8.4.2 Localizing along the Optical Axis 318 8.4.3 Brightness 320 8.4.4 Orientation 321 8.4.5 Color 322 8.5 Learning from Single-Molecule Signals 323 8.5.1 Determination of Molecular Associations 323 8.5.2 Determination of Molecular Conformations via FRET 325 8.5.3 Superresolution Single-Molecule Microscopy 329 8.5.4 Single-Molecule Tracking 332 8.5.5 Detecting Transitions 332 Acknowledgments 334 References 334 9 Super-Resolution Microscopy: Interference and Pattern Techniques 345 Gerrit Best, Roman Amberger, and Christoph Cremer 9.1 Introduction 345 9.1.1 Review: The Resolution Limit 346 9.2 Structured Illumination Microscopy (SIM) 347 9.2.1 Image Generation in Structured Illumination Microscopy 349 9.2.2 Extracting the High-Resolution Information 352 9.2.3 Optical Sectioning by SIM 353 9.2.4 How the Illumination Pattern is Generated 355 9.2.5 Mathematical Derivation of the Interference Pattern 355 9.2.6 Examples for SIM Setups 358 9.3 Spatially Modulated Illumination (SMI) Microscopy 362 9.3.1 Overview 362 9.3.2 SMI Setup 363 9.3.3 The Optical Path 364 9.3.4 Size Estimation with SMI Microscopy 366 9.4 Application of Patterned Techniques 368 9.5 Conclusion 372 9.6 Summary 372 Acknowledgments 373 References 373 10 STED Microscopy 375 Travis J. Gould, Patrina A. Pellett, and Joerg Bewersdorf 10.1 Introduction 375 10.2 The Concepts behind STED Microscopy 376 10.2.1 Fundamental Concepts 376 10.2.2 Key Parameters in STED Microscopy 380 10.3 Experimental Setup 384 10.3.1 Light Sources and Synchronization 384 10.3.2 Scanning and Speed 385 10.3.3 Multicolor STED Imaging 386 10.3.4 Improving Axial Resolution in STED Microscopy 388 10.4 Applications 388 10.4.1 Choice of Fluorophore 388 10.4.2 Labeling Strategies 389 Summary 390 References 391 A Appendix: Practical Guide to Optical Alignment 393 Rainer Heintzmann A.1 How to Obtain a Widened Parallel Laser Beam 393 A.2 Mirror Alignment 395 A.3 Lens Alignment 396 A.4 Autocollimation Telescope 396 A.5 Aligning a Single Lens Using a Laser Beam 397 A.6 How to Find the Focal Plane of a Lens 399 A.7 How to Focus to the Back Focal Plane of an Objective Lens 400 Index 403

About the Author :
Ulrich Kubitscheck is the department head of Biophysical Chemistry at the Institute of Physical and Theoretical Chemistry at the Rheinische Friedrich-Wilhelms-Universitat in Bonn, Germany. Having obtained his academic degrees from the University of Bremen, he spent his career working at The Weizmann Institute of Science and the Institute of Medical Physics and Biophysics of the Westfalische Wilhelms-Universitat Munster before taking up his present appointment at Bonn University. Professor Kubitscheck develops single molecule imaging techniques, has authored over 60 scientific publications and has extensive experience in teaching courses on physical chemistry, biophysics and quantitative microscopy.

Review :
“A companion website provides excellent material for anyone teaching a course on this topic.  Summing Up: Recommended.  Graduate students and researchers/faculty.”  (Choice, 1 January 2014) “This high-level textbook is best suited for advanced undergraduate and graduate students in the biosciences, and as a reference book for researchers in the field of high-performance microscopy.”  (Optics & Photonics News, 19 December 2013)