Diaspro Optical Fluorescence Microscopy

From the Spectral to the Nano Dimension
1. Auflage 2010
ISBN: 978-3-642-15175-0
Verlag: Springer
Format: PDF
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 244 Seiten

In the last decade, fluorescence microscopy has evolved from a classical “retrospective” microscopy approach into an advanced imaging technique that allows the observation of cellular activities in living cells with increased resolution and dimensions. A bright new future has arrived as the nano era has placed a whole new array of tools in the hands of biophysicists who are keen to go deeper into the intricacies of how biological systems work. Following an introduction to the complex world of optical microscopy, this book covers topics such as the concept of white confocal, nonlinear optical microscopy, fluctuation spectroscopies, site-specific labeling of proteins in living cells, imaging molecular physiology using nanosensors, measuring molecular dynamics, muscle braking and stem cell differentiation.

Autoren/Hrsg.

Diaspro, Alberto

Inhaltsverzeichnis

1;Preface;6
2;Contents;8
3;Contributors;10
4;Chapter 1: Fundamentals of Optical Microscopy;14
4.1;1.1 Introduction;14
4.1.1;1.1.1 The Human Visual System;14
4.1.2;1.1.2 History;15
4.1.3;1.1.3 The Basic Structure;16
4.2;1.2 Light;18
4.2.1;1.2.1 Ray Optics;18
4.2.2;1.2.2 Bright Field Microscopy;18
4.2.3;1.2.3 Köhler Illumination;19
4.2.4;1.2.4 The Spatial Frequency Plane;19
4.2.5;1.2.5 The Spatial Frequency Filtering;20
4.2.6;1.2.6 Digital Processing;21
4.2.7;1.2.7 Wave Optics;23
4.2.8;1.2.8 Interference;24
4.2.9;1.2.9 Phase Contrast;26
4.2.10;1.2.10 Differential Interference Contrast;28
4.2.11;1.2.11 Digital Holographic Microscopy;29
4.2.12;1.2.12 Polarization Contrast;31
4.2.13;1.2.13 Wavelength Contrast;32
4.2.14;1.2.14 Diffraction;34
4.3;1.3 Space;35
4.3.1;1.3.1 Field and Resolution;35
4.3.2;1.3.2 Field Extension;38
4.3.3;1.3.3 Resolution Enhancement;39
4.3.4;1.3.4 Resolution Enhancement Using Knowledge;41
4.3.5;1.3.5 Resolution Enhancement Using Matter;42
4.4;1.4 Time;44
4.4.1;1.4.1 Temporal Resolution;44
4.4.2;1.4.2 Duration;46
4.5;1.5 Conclusions;46
4.6;References;47
5;Chapter 2: The White Confocal: Continuous Spectral Tuning in Excitation and Emission;50
5.1;2.1 Fluorescence;50
5.1.1;2.1.1 Fluorescent Specimen;51
5.2;2.2 Fluorescence Microscopy;53
5.3;2.3 Confocal Fluorescence;54
5.4;2.4 A Tunable Laser;55
5.5;2.5 Tunable Beam Splitting;57
5.6;2.6 Tunable Spectral Detectors;59
5.7;2.7 Optimal Excitation;60
5.8;2.8 Optimal Excitation for Multiple Stainings;62
5.9;2.9 Förster Resonance Energy Transfer Problems;63
5.10;2.10 Excitation Spectra In Situ;65
5.11;2.11 .2-Maps;65
5.12;2.12 Fluorescence Lifetime Imaging;66
5.13;2.13 Unlimited Spectral Performance;66
5.14;References;66
6;Chapter 3: Second/Third Harmonic Generation Microscopy;68
6.1;3.1 Introduction;68
6.2;3.2 Nonlinear Optics Background;70
6.3;3.3 Second Harmonic Generation;73
6.3.1;3.3.1 SHG Microscopy;75
6.3.2;3.3.2 Applications;76
6.3.3;3.3.3 Polarization Dependence of SHG;77
6.4;3.4 Third Harmonic Generation;80
6.4.1;3.4.1 THG Microscopy;82
6.4.2;3.4.2 Applications;83
6.5;3.5 Laser Sources for SHG and THG Microscopy;84
6.6;3.6 Conclusion;84
6.7;References;85
7;Chapter 4: Role of Scattering and Nonlinear Effects in the Illumination and the Photobleaching Distribution Profiles;88
7.1;4.1 Introduction;88
7.2;4.2 Intensity Distribution of a Gaussian Beam;89
7.3;4.3 Intensity Distribution Modified by Scattering;90
7.4;4.4 Photobleaching Effects Induced by Scattering;92
7.5;4.5 Conclusions;96
7.6;References;96
8;Chapter 5: New Analytical Tools for Evaluation of Spherical Aberration in Optical Microscopy;98
8.1;5.1 Introduction;98
8.2;5.2 Basic Theory;99
8.3;5.3 Evolution of the Second-Order Moment;101
8.4;5.4 Generalized Second-Order Moment;103
8.5;5.5 Design of Beam-Shaping Elements for Reduction of SA Impact;105
8.6;5.6 Experimental Results;108
8.7;5.7 Conclusions;111
8.8;References;111
9;Chapter 6: Improving Image Formation by Pushing the Signal-to-Noise Ratio;113
9.1;6.1 Introduction;113
9.2;6.2 PSF and OTF;115
9.3;6.3 Pupil-Plane Filter Effects;117
9.4;6.4 Conclusion;121
9.5;References;121
10;Chapter 7: Site-Specific Labeling of Proteins in Living Cells Using Synthetic Fluorescent Dyes;123
10.1;7.1 Introduction;123
10.2;7.2 New Fluorescent Labels;124
10.2.1;7.2.1 Quantum Dots;124
10.2.2;7.2.2 Environmentally Sensitive Dyes;126
10.2.3;7.2.3 Photochromic Dyes;128
10.3;7.3 Site-Specific Chemical Labeling in Living Cells;130
10.3.1;7.3.1 Extracellular Chemical In Vivo-Labeling Techniques;130
10.3.1.1;7.3.1.1 Biotinylated Proteins as Chemical Handle for Labeling;130
10.3.1.2;7.3.1.2 Labeling of Carrier Protein Moieties - ACP and PCP;131
10.3.1.3;7.3.1.3 Sortagging: Sortase-Mediated Transpeptidation;132
10.3.2;7.3.2 Intracellular Chemical In Vivo-Labeling Techniques;133
10.3.2.1;7.3.2.1 Biarsenical-EDT2-Labeling;133
10.3.2.2;7.3.2.2 O6-Alkylguanine-DNA Alkyltransferase Labeling (AGT/SNAP Tag);135
10.3.2.3;7.3.2.3 HaloTag: Enzyme–Ligand Interaction Self-Labeling;136
10.4;7.4 In Vivo Labeling of Endogenous Proteins;136
10.5;7.5 Conclusion;138
10.6;References;139
11;Chapter 8: Imaging Molecular Physiology in Cells Using FRET-Based Fluorescent Nanosensors;143
11.1;8.1 Analytical Fluorescence Microscopy;143
11.1.1;8.1.1 Förster Resonance Energy Transfer;145
11.1.2;8.1.2 FRET Consequences;146
11.1.3;8.1.3 Lifetime Detection for FRET;149
11.2;8.2 Designing FRET-Based Biosensors;151
11.2.1;8.2.1 Reporters;151
11.2.2;8.2.2 Actuators;153
11.2.3;8.2.3 Multispecificity Detectors;153
11.2.3.1;8.2.3.1 Many Assays, Few Directions;154
11.2.3.2;8.2.3.2 Few Assays, Many Directions;155
11.2.3.3;8.2.3.3 Many Assays, Many Directions;155
11.2.4;8.2.4 Coincidence Detectors;156
11.3;8.3 Conclusion;161
11.4;References;161
12;Chapter 9: Measuring Molecular Dynamics by FRAP, FCS, and SPT;165
12.1;9.1 Introduction;165
12.2;9.2 Fluorescence Recovery After Photobleaching;165
12.3;9.3 Fluorescence Correlation Spectroscopy;168
12.4;9.4 Single Particle Tracking;169
12.5;9.5 Conclusion;171
12.6;References;171
13;Chapter 10: In Vitro–In Vivo Fluctuation Spectroscopies;176
13.1;10.1 Introduction;176
13.2;10.2 Fluctuation Spectroscopy: General Principles;177
13.2.1;10.2.1 Average Fluctuations of the Fluorescence Signal;177
13.2.2;10.2.2 ACF in a Generic Optical Field;178
13.2.3;10.2.3 Generalized Excitation Modes;180
13.2.3.1;10.2.3.1 Dual Beam Excitation: ACF;181
13.2.3.2;10.2.3.2 Dual Beam Excitation: CCF;181
13.2.3.3;10.2.3.3 Scanning FCS;182
13.2.3.4;10.2.3.4 Chemical Kinetics;184
13.3;10.3 Experimental Examples;185
13.3.1;10.3.1 In Vitro Experiments: Photodynamics of Fluorescent Proteins Trapped in Agarose Gels;185
13.3.2;10.3.2 In Vivo Experiments: Nanoparticles Targeting of Cells, Tracking and Fluctuations;189
13.4;10.4 Conclusions;191
13.5;References;191
14;Chapter 11: Interference X-ray Diffraction from Single Muscle Cells Reveals the Molecular Basis of Muscle Braking;193
14.1;11.1 Introduction;193
14.2;11.2 Experimental Protocol and Results;195
14.3;11.3 Conclusions;198
14.4;References;198
15;Chapter 12: Low Concentration Protein Detection Using Novel SERS Devices;200
15.1;12.1 Introduction;200
15.2;12.2 Experimental;202
15.2.1;12.2.1 Device Fabrication;202
15.2.1.1;12.2.1.1 Periodic Gold Nanoarray SERS Device (``Device1´´);202
15.2.1.2;12.2.1.2 Site Selective Electroless SERS Device (``Device2´´);202
15.2.2;12.2.2 Sample Preparation;203
15.2.3;12.2.3 Characterization Technique;204
15.2.4;12.2.4 Data Analysis;205
15.3;12.3 Results and Discussions;206
15.3.1;12.3.1 Proteins on ``Device1´´;206
15.3.1.1;12.3.1.1 Bovine Serum Albumin;207
15.3.1.2;12.3.1.2 Lysozyme;210
15.3.1.3;12.3.1.3 Ribonuclease-B;212
15.3.1.4;12.3.1.4 Ferritin;214
15.3.2;12.3.2 Rhodamine 6G (R6G) on ``Device2´´;216
15.4;12.4 Conclusions;217
15.5;References;217
16;Chapter 13: Near Infrared Three-Dimensional Nonlinear Optical Monitoring of Stem Cell Differentiation;220
16.1;13.1 Introduction;220
16.1.1;13.1.1 Stem Cells;220
16.1.2;13.1.2 Differentiation into Chondrocytes;221
16.1.3;13.1.3 Differentiation into Neurons;222
16.1.4;13.1.4 Differentiation into Pancreatic Cells;222
16.1.5;13.1.5 Nonlinear Optical/Second Harmonic Generation Imaging;223
16.2;13.2 Materials and Methods;224
16.2.1;13.2.1 Cell Culture;224
16.2.2;13.2.2 Hanging Drop Cultures and Induction of Chondrogenic/Pancreatic Differentiation;224
16.2.3;13.2.3 Induction of Neuronal Differentiation;226
16.2.4;13.2.4 Immunohistochemical Localisations;226
16.2.5;13.2.5 Imaging and 3D Monitoring;227
16.2.5.1;13.2.5.1 High-Resolution Two/Multiphoton Imaging;227
16.2.5.2;13.2.5.2 Image Processing and Analysis;227
16.3;13.3 Results;228
16.3.1;13.3.1 Evidence for Two-Photon Excitation (TPE) and Second Harmonic Generation (SHG);228
16.3.2;13.3.2 Cell Morphology and Organization of Fibrillar Collagen;229
16.3.2.1;13.3.2.1 Attached Cartilaginous Embryoid Bodies (2D Cultures);229
16.3.2.2;13.3.2.2 Attached Pancreatic Embryoid Bodies (2D Cultures);229
16.3.2.3;13.3.2.3 Embryoid Bodies in 3D Scaffold;230
16.3.3;13.3.3 Immunolocalization Studies;231
16.3.3.1;13.3.3.1 Nanog;231
16.3.3.2;13.3.3.2 Attached Neuronal Embryoid Bodies (2D Cultures);232
16.3.3.3;13.3.3.3 Collagen II;232
16.3.4;13.3.4 Forward and Backward Second Harmonic Generation Signals;233
16.4;13.4 Discussion;233
16.4.1;13.4.1 Chondrogenic Nodules;234
16.5;13.5 Conclusions;235
16.6;References;236
17;Chapter 14: A Correlative Microscopy: A Combination of Light and Electron Microscopy;239
17.1;14.1 Introduction;239
17.2;14.2 Classical CLEM Approaches;241
17.3;14.3 Cryo-CLEM Approaches;243
17.4;References;245
18;Index;247