Fluorescence microscopy : from principles to biological applications / edited by Ulrich Kubitscheck.

Format:

Book

Contributor:

Kubitscheck, Ulrich, editor.

Wiley InterScience (Online service)

Rudolph G. Schmieder Fund.

Language:

English

Subjects (All):

Fluorescence microscopy.

Physical Description:

1 online resource (xx, 410 pages) : illustrations

Place of Publication:

Weinheim : Wiley-Blackwell, [2013].

System Details:

text file

Summary:

A comprehensive introduction to advanced fluorescence microscopy methods and their applications. This is the first title on the topic designed specifically to allow students and researchers with little background in physics to understand both microscopy basics and novel light microscopy techniques. The book is written by renowned experts and pioneers in the field with a rather intuitive than formal approach. It always keeps the non-expert reader in mind, making even unavoidable complex theoretical concepts readily accessible. All commonly used methods are covered. A companion website with additional references, examples and video material makes this a valuable teaching resource: Book jacket.

Contents:

1 Introduction to Optics and Photophysics / Rainer Heintzmann Heintzmann, Rainer 1

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 / Ulrich Kubitscheck Kubitscheck, Ulrich 33

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 Fraction 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 / Jurek W. Dobrucki Dobrucki, Jurek W. 97

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 Photobleadung 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 / Gerd Ulrich Nienhaus Nienhaus, Gerd Ulrich, Karin Nienhaus Nienhaus, Karin 143

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 Nanopartides 150

4.4.3 Conjugation Strategies for Synthetic Fluorophores 152

4.4.4 Nonnatural Amino Adds 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 / Nikolaus Naredi-Rainer Naredi-Rainer, Nikolaus, Jens Prescher Prescher, Jens, Achim Hartschuh Hartschuh, Achim, Don C. Lamb Lamb, Don C. 175

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 Nonscanrring 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 / Reiner Peters Peters, Reiner 215

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 / Fred S. Wouters Wouters, Fred S. 245

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 / Markus Axmann Axmann, Markus, Josef Madl Madl, Josef, Gerhard J. Schütz Schütz, Gerhard J. 293

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 Superrcsolution 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 / Gerrit Best Best, Gerrit, Roman Amberger Amberger, Roman, Christoph Cremer Cremer, Christoph 345

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 / Travis J. Gould Gould, Travis J., Patrina A. Pellett Pellett, Patrina A., Joerg Bewersdorf Bewersdorf, Joerg 375

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.

Notes:

Description based on print version record.

Includes bibliographic references and index.

Electronic reproduction. Hoboken, N.J. Available via World Wide Web.

Local Notes:

Acquired for the Penn Libraries with assistance from the Rudolph G. Schmieder Fund.

ISBN:

3527671609

9783527671601

Publisher Number:

99958071772

Access Restriction:

Restricted for use by site license.