Title Details

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 Münster, 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.

List of Contributors xv

Preface xix

1 Introduction to Optics 1
Rainer Heintzmann and Ulrich Kubitscheck

1.1 A Short History of Theories about Light 1

1.2 Properties of LightWaves 2

1.3 Four Effects of Interference 7

1.4 Optical Elements 13

1.5 Optical Aberrations 20

2 Principles of LightMicroscopy 23
Ulrich Kubitscheck

2.1 Introduction 23

2.2 Construction of Light Microscopes 23

2.3 Wave Optics and Resolution 32

2.4 Apertures, Pupils, and Telecentricity 50

2.5 Microscope Objectives 53

2.6 Contrast 67

2.7 Summary 82

3 Fluorescence Microscopy 85
JurekW. Dobrucki and Ulrich Kubitscheck

3.1 Contrast in Optical Microscopy 85

3.2 Physical Foundations of Fluorescence 86

3.3 Features of Fluorescence Microscopy 90

3.4 A Fluorescence Microscope 95

3.5 Types ofNoise in a Digital Microscopy Image 114

3.6 Quantitative Fluorescence Microscopy 119

3.7 Limitations of Fluorescence Microscopy 124

3.8 Summary and Outlook 128

4 Fluorescence Labeling 133
Gerd Ulrich Nienhaus and Karin Nienhaus

4.1 Introduction 133

4.2 Key Properties of Fluorescent Labels 133

4.3 Synthetic Fluorophores 138

4.4 Genetically Encoded Labels 149

4.5 Label Selection for Particular Applications 155

5 Confocal Microscopy 165
Nikolaus Naredi-Rainer, Jens Prescher, Achim Hartschuh, and Don C. Lamb

5.1 Evolution and Limits of ConventionalWidefield Microscopy 165

5.2 Theory of Confocal Microscopy 166

5.3 Applications of Confocal Microscopy 186

6 Two-Photon Excitation Microscopy for Three-Dimensional Imaging of Living Intact Tissues 203
David W. Piston

6.1 Introduction 203

6.2 What is Two-Photon Excitation? 205

6.3 How Does Two-Photon Excitation MicroscopyWork in Practice? 211

6.4 Instrumentation 216

6.5 Limitations of Two-Photon Excitation Microscopy 222

6.6 When is 2PMthe Best Option? 229

6.7 Applications of Two-Photon Microscopy 231

6.8 Other NonlinearMicroscopies 239

6.9 Future Outlook for 2PM 240

7 Light Sheet Microscopy 243
Gopi Shah,MichaelWeber, and Jan Huisken

7.1 Principle of Light Sheet Microscopy 244

7.2 Light Sheet Microscopy: Key Advantages 245

7.3 Construction andWorking of a Light Sheet Microscope 246

7.4 Theory of Light Sheet Microscopy 247

7.5 Light Sheet Interaction with Tissue 251

7.6 3D Imaging 253

7.7 Multiview Imaging 255

7.8 Different Lens Configurations 257

7.9 Sample Mounting 258

7.10 Recent Advances in Light Sheet Microscopy 259

7.11 Outlook 260

8 Localization-Based Super-Resolution Microscopy 267
Markus Sauer and Mike Heilemann

8.1 Super-Resolution Microscopy: An Introduction 267

8.2 The Principle of Single-Molecule Localization Microscopy 269

8.3 Photoactivatable and Photoconvertible Probes 272

8.4 Intrinsically Photoswitchable Probes 272

8.5 Photoswitching of Organic Fluorophores by Chemical Reactions 273

8.6 Experimental Setup for Localization Microscopy 273

8.7 Optical Resolution and Imaging Artifacts 276

8.8 Fluorescence Labeling for Super-Resolution Microscopy 278

8.9 Measures for Improving Imaging Contrast 283

8.10 SMLM Software 283

8.11 Reference Structures for SMLM 285

8.12 Quantification of SMLM Data 286

9 Super-Resolution Microscopy: Interference and Pattern Techniques 291
Udo Birk, Gerrit Best, Roman Amberger, and Christoph Cremer

9.1 Introduction 291

9.2 Structured Illumination Microscopy (SIM) 293

9.3 SpatiallyModulated Illumination (SMI) Microscopy 307

9.4 Application of Patterned Techniques 313

10 STEDMicroscopy 321
Travis J. Gould, Lena K. Schroeder, Patrina A. Pellett, and Joerg Bewersdorf

10.1 Introduction 321

10.2 The Concepts behind STED Microscopy 322

10.3 Experimental Setup 330

10.4 Applications 334

11 Fluorescence Photobleaching Techniques 339
Reiner Peters

11.1 Introduction 339

11.2 Basic Concepts and Procedures 340

11.3 Fluorescence Recovery after Photobleaching (FRAP) 345

11.4 Continuous Fluorescence Microphotolysis (CFM) 352

11.5 CLSM-Assisted Photobleaching Methods 356

12 Single-Molecule Microscopy in the Life Sciences 365
Markus Axmann, JosefMadl, and Gerhard J. Schütz

12.1 Encircling the Problem 365

12.2 What is the Unique Information? 367

12.3 Building a Single-Molecule Microscope 372

12.4 Analyzing Single-Molecule Signals: Position, Orientation, Color, and Brightness 387

12.5 Learning from Single-Molecule Signals 394

13 Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging 405
Fred S.Wouters

13.1 General Introduction 405

13.2 Förster Resonance Energy Transfer 406

13.3 Measuring FRET 426

13.4 FLIM 439

13.5 Analysis and Pitfalls 444

A Appendix A:What Exactly is a Digital Image? 453
Ulrich Kubitscheck

A.1 Introduction 453

A.2 Digital Images as Matrices 453

A.3 Look-up Table 457

A.4 Intensity Histograms 457

A.5 Image Processing 458

A.6 Pitfalls 460

B Appendix B: Practical Guide to Optical Alignment 463
Rainer Heintzmann

B.1 How to Obtain aWidened Parallel Laser Beam? 463

B.2 Mirror Alignment 465

B.3 Lens Alignment 466

B.4 Autocollimation Telescope 466

B.5 Aligning a Single Lens Using a Laser Beam 466

B.6 How to Find the Focal Plane of a Lens? 469

B.7 How to Focus to the Back Focal Plane of an Objective Lens? 470

Index 473