Optical Fluorescence Microscopy (eBook)

Preface 6
Contents 8
Contributors 10
Chapter 1: Fundamentals of Optical Microscopy 14
1.1 Introduction 14
1.1.1 The Human Visual System 14
1.1.2 History 15
1.1.3 The Basic Structure 16
1.2 Light 18
1.2.1 Ray Optics 18
1.2.2 Bright Field Microscopy 18
1.2.3 Köhler Illumination 19
1.2.4 The Spatial Frequency Plane 19
1.2.5 The Spatial Frequency Filtering 20
1.2.6 Digital Processing 21
1.2.7 Wave Optics 23
1.2.8 Interference 24
1.2.9 Phase Contrast 26
1.2.10 Differential Interference Contrast 28
1.2.11 Digital Holographic Microscopy 29
1.2.12 Polarization Contrast 31
1.2.13 Wavelength Contrast 32
1.2.14 Diffraction 34
1.3 Space 35
1.3.1 Field and Resolution 35
1.3.2 Field Extension 38
1.3.3 Resolution Enhancement 39
1.3.4 Resolution Enhancement Using Knowledge 41
1.3.5 Resolution Enhancement Using Matter 42
1.4 Time 44
1.4.1 Temporal Resolution 44
1.4.2 Duration 46
1.5 Conclusions 46
References 47
Chapter 2: The White Confocal: Continuous Spectral Tuning in Excitation and Emission 50
2.1 Fluorescence 50
2.1.1 Fluorescent Specimen 51
2.2 Fluorescence Microscopy 53
2.3 Confocal Fluorescence 54
2.4 A Tunable Laser 55
2.5 Tunable Beam Splitting 57
2.6 Tunable Spectral Detectors 59
2.7 Optimal Excitation 60
2.8 Optimal Excitation for Multiple Stainings 62
2.9 Förster Resonance Energy Transfer Problems 63
2.10 Excitation Spectra In Situ 65
2.11 .2-Maps 65
2.12 Fluorescence Lifetime Imaging 66
2.13 Unlimited Spectral Performance 66
References 66
Chapter 3: Second/Third Harmonic Generation Microscopy 68
3.1 Introduction 68
3.2 Nonlinear Optics Background 70
3.3 Second Harmonic Generation 73
3.3.1 SHG Microscopy 75
3.3.2 Applications 76
3.3.3 Polarization Dependence of SHG 77
3.4 Third Harmonic Generation 80
3.4.1 THG Microscopy 82
3.4.2 Applications 83
3.5 Laser Sources for SHG and THG Microscopy 84
3.6 Conclusion 84
References 85
Chapter 4: Role of Scattering and Nonlinear Effects in the Illumination and the Photobleaching Distribution Profiles 88
4.1 Introduction 88
4.2 Intensity Distribution of a Gaussian Beam 89
4.3 Intensity Distribution Modified by Scattering 90
4.4 Photobleaching Effects Induced by Scattering 92
4.5 Conclusions 96
References 96
Chapter 5: New Analytical Tools for Evaluation of Spherical Aberration in Optical Microscopy 98
5.1 Introduction 98
5.2 Basic Theory 99
5.3 Evolution of the Second-Order Moment 101
5.4 Generalized Second-Order Moment 103
5.5 Design of Beam-Shaping Elements for Reduction of SA Impact 105
5.6 Experimental Results 108
5.7 Conclusions 111
References 111
Chapter 6: Improving Image Formation by Pushing the Signal-to-Noise Ratio 113
6.1 Introduction 113
6.2 PSF and OTF 115
6.3 Pupil-Plane Filter Effects 117
6.4 Conclusion 121
References 121
Chapter 7: Site-Specific Labeling of Proteins in Living Cells Using Synthetic Fluorescent Dyes 123
7.1 Introduction 123
7.2 New Fluorescent Labels 124
7.2.1 Quantum Dots 124
7.2.2 Environmentally Sensitive Dyes 126
7.2.3 Photochromic Dyes 128
7.3 Site-Specific Chemical Labeling in Living Cells 130
7.3.1 Extracellular Chemical In Vivo-Labeling Techniques 130
7.3.1.1 Biotinylated Proteins as Chemical Handle for Labeling 130
7.3.1.2 Labeling of Carrier Protein Moieties - ACP and PCP 131
7.3.1.3 Sortagging: Sortase-Mediated Transpeptidation 132
7.3.2 Intracellular Chemical In Vivo-Labeling Techniques 133
7.3.2.1 Biarsenical-EDT2-Labeling 133
7.3.2.2 O6-Alkylguanine-DNA Alkyltransferase Labeling (AGT/SNAP Tag) 135
7.3.2.3 HaloTag: Enzyme–Ligand Interaction Self-Labeling 136
7.4 In Vivo Labeling of Endogenous Proteins 136
7.5 Conclusion 138
References 139
Chapter 8: Imaging Molecular Physiology in Cells Using FRET-Based Fluorescent Nanosensors 143
8.1 Analytical Fluorescence Microscopy 143
8.1.1 Förster Resonance Energy Transfer 145
8.1.2 FRET Consequences 146
8.1.3 Lifetime Detection for FRET 149
8.2 Designing FRET-Based Biosensors 151
8.2.1 Reporters 151
8.2.2 Actuators 153
8.2.3 Multispecificity Detectors 153
8.2.3.1 Many Assays, Few Directions 154
8.2.3.2 Few Assays, Many Directions 155
8.2.3.3 Many Assays, Many Directions 155
8.2.4 Coincidence Detectors 156
8.3 Conclusion 161
References 161
Chapter 9: Measuring Molecular Dynamics by FRAP, FCS, and SPT 165
9.1 Introduction 165
9.2 Fluorescence Recovery After Photobleaching 165
9.3 Fluorescence Correlation Spectroscopy 168
9.4 Single Particle Tracking 169
9.5 Conclusion 171
References 171
Chapter 10: In Vitro–In Vivo Fluctuation Spectroscopies 176
10.1 Introduction 176
10.2 Fluctuation Spectroscopy: General Principles 177
10.2.1 Average Fluctuations of the Fluorescence Signal 177
10.2.2 ACF in a Generic Optical Field 178
10.2.3 Generalized Excitation Modes 180
10.2.3.1 Dual Beam Excitation: ACF 181
10.2.3.2 Dual Beam Excitation: CCF 181
10.2.3.3 Scanning FCS 182
10.2.3.4 Chemical Kinetics 184
10.3 Experimental Examples 185
10.3.1 In Vitro Experiments: Photodynamics of Fluorescent Proteins Trapped in Agarose Gels 185
10.3.2 In Vivo Experiments: Nanoparticles Targeting of Cells, Tracking and Fluctuations 189
10.4 Conclusions 191
References 191
Chapter 11: Interference X-ray Diffraction from Single Muscle Cells Reveals the Molecular Basis of Muscle Braking 193
11.1 Introduction 193
11.2 Experimental Protocol and Results 195
11.3 Conclusions 198
References 198
Chapter 12: Low Concentration Protein Detection Using Novel SERS Devices 200
12.1 Introduction 200
12.2 Experimental 202
12.2.1 Device Fabrication 202
12.2.1.1 Periodic Gold Nanoarray SERS Device (``Device1´´) 202
12.2.1.2 Site Selective Electroless SERS Device (``Device2´´) 202
12.2.2 Sample Preparation 203
12.2.3 Characterization Technique 204
12.2.4 Data Analysis 205
12.3 Results and Discussions 206
12.3.1 Proteins on ``Device1´´ 206
12.3.1.1 Bovine Serum Albumin 207
12.3.1.2 Lysozyme 210
12.3.1.3 Ribonuclease-B 212
12.3.1.4 Ferritin 214
12.3.2 Rhodamine 6G (R6G) on ``Device2´´ 216
12.4 Conclusions 217
References 217
Chapter 13: Near Infrared Three-Dimensional Nonlinear Optical Monitoring of Stem Cell Differentiation 220
13.1 Introduction 220
13.1.1 Stem Cells 220
13.1.2 Differentiation into Chondrocytes 221
13.1.3 Differentiation into Neurons 222
13.1.4 Differentiation into Pancreatic Cells 222
13.1.5 Nonlinear Optical/Second Harmonic Generation Imaging 223
13.2 Materials and Methods 224
13.2.1 Cell Culture 224
13.2.2 Hanging Drop Cultures and Induction of Chondrogenic/Pancreatic Differentiation 224
13.2.3 Induction of Neuronal Differentiation 226
13.2.4 Immunohistochemical Localisations 226
13.2.5 Imaging and 3D Monitoring 227
13.2.5.1 High-Resolution Two/Multiphoton Imaging 227
13.2.5.2 Image Processing and Analysis 227
13.3 Results 228
13.3.1 Evidence for Two-Photon Excitation (TPE) and Second Harmonic Generation (SHG) 228
13.3.2 Cell Morphology and Organization of Fibrillar Collagen 229
13.3.2.1 Attached Cartilaginous Embryoid Bodies (2D Cultures) 229
13.3.2.2 Attached Pancreatic Embryoid Bodies (2D Cultures) 229
13.3.2.3 Embryoid Bodies in 3D Scaffold 230
13.3.3 Immunolocalization Studies 231
13.3.3.1 Nanog 231
13.3.3.2 Attached Neuronal Embryoid Bodies (2D Cultures) 232
13.3.3.3 Collagen II 232
13.3.4 Forward and Backward Second Harmonic Generation Signals 233
13.4 Discussion 233
13.4.1 Chondrogenic Nodules 234
13.5 Conclusions 235
References 236
Chapter 14: A Correlative Microscopy: A Combination of Light and Electron Microscopy 239
14.1 Introduction 239
14.2 Classical CLEM Approaches 241
14.3 Cryo-CLEM Approaches 243
References 245
Index 247