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<p>List of Contributors xv</p> <p>Preface xix</p> <p><b>1 Introduction to Optics 1</b><br /><i>Rainer Heintzmann and Ulrich Kubitscheck</i></p> <p>1.1 A Short History of Theories about Light 1</p> <p>1.2 Properties of LightWaves 2</p> <p>1.3 Four Effects of Interference 7</p> <p>1.4 Optical Elements 13</p> <p>1.5 Optical Aberrations 20</p> <p><b>2 Principles of LightMicroscopy 23</b><br /><i>Ulrich Kubitscheck</i></p> <p>2.1 Introduction 23</p> <p>2.2 Construction of Light Microscopes 23</p> <p>2.3 Wave Optics and Resolution 32</p> <p>2.4 Apertures, Pupils, and Telecentricity 50</p> <p>2.5 Microscope Objectives 53</p> <p>2.6 Contrast 67</p> <p>2.7 Summary 82</p> <p><b>3 Fluorescence Microscopy 85</b><br /><i>JurekW. Dobrucki and Ulrich Kubitscheck</i></p> <p>3.1 Contrast in Optical Microscopy 85</p> <p>3.2 Physical Foundations of Fluorescence 86</p> <p>3.3 Features of Fluorescence Microscopy 90</p> <p>3.4 A Fluorescence Microscope 95</p> <p>3.5 Types ofNoise in a Digital Microscopy Image 114</p> <p>3.6 Quantitative Fluorescence Microscopy 119</p> <p>3.7 Limitations of Fluorescence Microscopy 124</p> <p>3.8 Summary and Outlook 128</p> <p><b>4 Fluorescence Labeling 133</b><br /><i>Gerd Ulrich Nienhaus and Karin Nienhaus</i></p> <p>4.1 Introduction 133</p> <p>4.2 Key Properties of Fluorescent Labels 133</p> <p>4.3 Synthetic Fluorophores 138</p> <p>4.4 Genetically Encoded Labels 149</p> <p>4.5 Label Selection for Particular Applications 155</p> <p><b>5 Confocal Microscopy 165</b><br /><i>Nikolaus Naredi-Rainer, Jens Prescher, Achim Hartschuh, and Don C. Lamb</i></p> <p>5.1 Evolution and Limits of ConventionalWidefield Microscopy 165</p> <p>5.2 Theory of Confocal Microscopy 166</p> <p>5.3 Applications of Confocal Microscopy 186</p> <p><b>6 Two-Photon Excitation Microscopy for Three-Dimensional Imaging of Living Intact Tissues 203</b><br /><i>David W. Piston</i></p> <p>6.1 Introduction 203</p> <p>6.2 What is Two-Photon Excitation? 205</p> <p>6.3 How Does Two-Photon Excitation MicroscopyWork in Practice? 211</p> <p>6.4 Instrumentation 216</p> <p>6.5 Limitations of Two-Photon Excitation Microscopy 222</p> <p>6.6 When is 2PMthe Best Option? 229</p> <p>6.7 Applications of Two-Photon Microscopy 231</p> <p>6.8 Other NonlinearMicroscopies 239</p> <p>6.9 Future Outlook for 2PM 240</p> <p><b>7 Light Sheet Microscopy 243</b><br /><i>Gopi Shah,MichaelWeber, and Jan Huisken</i></p> <p>7.1 Principle of Light Sheet Microscopy 244</p> <p>7.2 Light Sheet Microscopy: Key Advantages 245</p> <p>7.3 Construction andWorking of a Light Sheet Microscope 246</p> <p>7.4 Theory of Light Sheet Microscopy 247</p> <p>7.5 Light Sheet Interaction with Tissue 251</p> <p>7.6 3D Imaging 253</p> <p>7.7 Multiview Imaging 255</p> <p>7.8 Different Lens Configurations 257</p> <p>7.9 Sample Mounting 258</p> <p>7.10 Recent Advances in Light Sheet Microscopy 259</p> <p>7.11 Outlook 260</p> <p><b>8 Localization-Based Super-Resolution Microscopy 267</b><br /><i>Markus Sauer and Mike Heilemann</i></p> <p>8.1 Super-Resolution Microscopy: An Introduction 267</p> <p>8.2 The Principle of Single-Molecule Localization Microscopy 269</p> <p>8.3 Photoactivatable and Photoconvertible Probes 272</p> <p>8.4 Intrinsically Photoswitchable Probes 272</p> <p>8.5 Photoswitching of Organic Fluorophores by Chemical Reactions 273</p> <p>8.6 Experimental Setup for Localization Microscopy 273</p> <p>8.7 Optical Resolution and Imaging Artifacts 276</p> <p>8.8 Fluorescence Labeling for Super-Resolution Microscopy 278</p> <p>8.9 Measures for Improving Imaging Contrast 283</p> <p>8.10 SMLM Software 283</p> <p>8.11 Reference Structures for SMLM 285</p> <p>8.12 Quantification of SMLM Data 286</p> <p><b>9 Super-Resolution Microscopy: Interference and Pattern Techniques 291</b><br /><i>Udo Birk, Gerrit Best, Roman Amberger, and Christoph Cremer</i></p> <p>9.1 Introduction 291</p> <p>9.2 Structured Illumination Microscopy (SIM) 293</p> <p>9.3 SpatiallyModulated Illumination (SMI) Microscopy 307</p> <p>9.4 Application of Patterned Techniques 313</p> <p><b>10 STEDMicroscopy 321</b><br /><i>Travis J. Gould, Lena K. Schroeder, Patrina A. Pellett, and Joerg Bewersdorf</i></p> <p>10.1 Introduction 321</p> <p>10.2 The Concepts behind STED Microscopy 322</p> <p>10.3 Experimental Setup 330</p> <p>10.4 Applications 334</p> <p><b>11 Fluorescence Photobleaching Techniques 339</b><br /><i>Reiner Peters</i></p> <p>11.1 Introduction 339</p> <p>11.2 Basic Concepts and Procedures 340</p> <p>11.3 Fluorescence Recovery after Photobleaching (FRAP) 345</p> <p>11.4 Continuous Fluorescence Microphotolysis (CFM) 352</p> <p>11.5 CLSM-Assisted Photobleaching Methods 356</p> <p><b>12 Single-Molecule Microscopy in the Life Sciences 365</b><br /><i>Markus Axmann, JosefMadl, and Gerhard J. Schütz</i></p> <p>12.1 Encircling the Problem 365</p> <p>12.2 What is the Unique Information? 367</p> <p>12.3 Building a Single-Molecule Microscope 372</p> <p>12.4 Analyzing Single-Molecule Signals: Position, Orientation, Color, and Brightness 387</p> <p>12.5 Learning from Single-Molecule Signals 394</p> <p><b>13 Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging 405</b><br /><i>Fred S.Wouters</i></p> <p>13.1 General Introduction 405</p> <p>13.2 Förster Resonance Energy Transfer 406</p> <p>13.3 Measuring FRET 426</p> <p>13.4 FLIM 439</p> <p>13.5 Analysis and Pitfalls 444</p> <p>A Appendix A:What Exactly is a Digital Image? 453<br /><i>Ulrich Kubitscheck</i></p> <p>A.1 Introduction 453</p> <p>A.2 Digital Images as Matrices 453</p> <p>A.3 Look-up Table 457</p> <p>A.4 Intensity Histograms 457</p> <p>A.5 Image Processing 458</p> <p>A.6 Pitfalls 460</p> <p>B Appendix B: Practical Guide to Optical Alignment 463<br /><i>Rainer Heintzmann</i></p> <p>B.1 How to Obtain aWidened Parallel Laser Beam? 463</p> <p>B.2 Mirror Alignment 465</p> <p>B.3 Lens Alignment 466</p> <p>B.4 Autocollimation Telescope 466</p> <p>B.5 Aligning a Single Lens Using a Laser Beam 466</p> <p>B.6 How to Find the Focal Plane of a Lens? 469</p> <p>B.7 How to Focus to the Back Focal Plane of an Objective Lens? 470</p> <p>Index 473</p>

Ulrich Kubitscheck is the department head of Biophysical Chemistry at the Institute of Physical and Theoretical Chemistry at the University of Bonn, Germany. Having obtained his academic degrees from the University of Bremen, Germany, he spent his career working at The Weizmann Institute of Science and the Institute of Medical Physics and Biophysics of the University of Munster, Germany, before taking up his present appointment at the University of Bonn. Professor Kubitscheck develops single molecule and light sheet imaging techniques, has authored over 80 scientific publications and has extensive experience in teaching courses on physical chemistry, biophysics and quantitative microscopy.

This text is specifically designed to allow advanced students and researchers with little background in physics to comprehend both microscopy basics and state-of the-art light microscopy techniques. This second edition was carefully revised and features new chapters on super resolution, 2-photon and light sheet fluorescence microscopy, making it even more suitable for teaching and training courses on modern microscopy. Each chapter is written by a renowned expert in the field, and has been thoroughly revised to include more didactical features: Theoretical derivations have been concentrated in text boxes and summaries provide fast access for beginners. The supplementary material has been expanded, with a companion website that offers additional teaching material including videos.

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