Gadella FRET and FLIM Techniques

1. Auflage 2011
ISBN: 978-0-08-091512-8
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 560 Seiten, Web PDF

This volume reviews the techniques Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) providing researchers with step by step protocols and handy hints and tips. Both have become staple techniques in many biological and biophysical fields.

Autoren/Hrsg.

Gadella, Theodorus W. J.

Inhaltsverzeichnis

1;Front Cover;4
2;Fret and Flim Techniques;4
3;Copyright Page;5
4;Contents;6
5;Contributors;12
6;Preface;18
7;Chapter 1: Foumlrster resonance energy transfer-FRET what is it, why do it, and how it's done;26
7.1;1.1. Introduction;26
7.2;1.2. Historical background; setting the groundwork;29
7.3;1.3. FRET basics;35
7.4;1.4. What can we learn from energy transfer?;47
7.5;1.5. Simple depiction of basic fluorescence; how you determine the FRET efficiency;49
7.6;1.6. Measuring fluorescence emission of the donor;59
7.7;1.7. Measuring any de-excitation process in order to determine FRET efficiency; the acronym FRET;63
7.8;1.8. Measuring photobleaching to determine FRET efficiency;65
7.9;1.9. Measuring the acceptor fluorescence or reaction products to determine the efficiency of energy transfer;67
7.10;1.10. Transfer between identical molecules: Fluorescence anisotropy;71
7.11;1.11. Energy transfer by electron exchange;72
7.12;Acknowledgments;73
7.13;References;73
8;Chapter 2: Frequency domain FLIM theory, instrumentation, and data analysis;84
8.1;2.1. Rates, time constants, and lifetimes;86
8.2;2.2. Instrumentation;91
8.3;2.3. Instrumentation: Frequency domain FLIM;93
8.4;2.4. Systems for measuring lifetimes at multiple frequencies;95
8.5;2.5. Spectral FLIM;95
8.6;2.6. Data acquisition strategies;97
8.7;2.7. Calibration and measurement validation;98
8.8;2.8. Calibration by comparison with a scattering solution;98
8.9;2.9. Calibration by use of reflecting surfaces;99
8.10;2.10. Calibration by use of fluorophores of known lifetime;100
8.11;2.11. Comparison of calibration methods;100
8.12;2.12. Validation after calibration;101
8.13;2.13. First pass analysis-data to modulation depth and phase shift;102
8.14;2.14. Fourier methods for estimating phase and modulation;102
8.15;2.15. Sine fitting methods for estimating phase and modulation;104
8.16;2.16. Two-component analysis of FLIM data;105
8.17;2.17. Application: Semi-quantitative FRET analysis;109
8.18;2.18. Application: Quantitative FRET analysis;111
8.19;2.19. Emerging techniques;113
8.20;2.20. Segmented intensifiers;113
8.21;2.21. Directly modulated detection schemes;114
8.22;Acknowledgments;115
8.23;References;115
9;Chapter 3: Time domain FLIM: Theory, instrumentation, and data analysis;120
9.1;3.1. Introduction;121
9.2;3.2. Lifetime detection methods;122
9.3;3.3. Point scanning time domain FLIM implementations;129
9.4;3.4. Wide field time-domain FLIM implementations;134
9.5;3.5. Signal considerations and limitations;137
9.6;3.6. Data analysis;147
9.7;3.7. FRET-FLIM example application;150
9.8;References;153
10;Chapter 4: Multidimensional fluorescence imaging;158
10.1;4.1. Introduction;159
10.2;4.2. FLIM and wide-field time-gated imaging;162
10.3;4.3. Spectrally resolved FLIM;169
10.4;4.4. Polarization-resolved imaging;180
10.5;4.5. Conclusions;185
10.6;Acknowledgments;187
10.7;References;187
11;Chapter 5: Visible fluorescent proteins for FRET;196
11.1;5.1. Introduction;197
11.2;5.2. Fluorescent proteins;198
11.3;5.3. Variants of avGFP;203
11.4;5.4. Variants of DsRed;209
11.5;5.5. Variants of eqFP578 and eqFP611;210
11.6;5.6. VFP variants from other sources;211
11.7;5.7. Chromoproteins and fluorescent derivatives;212
11.8;5.8. Optical highlighter fluorescent proteins;213
11.9;5.9. Choosing the right VFP FRET pair;215
11.10;5.10. VFP-based FRET pairs;221
11.11;5.11. Applications;228
11.12;5.12. Conclusion;233
11.13;Acknowledgments;234
11.14;References;234
12;Chapter 6: Small molecule-based FRET probes;250
12.1;6.1. Introduction;250
12.2;6.2. Fluorophores and quenchers;251
12.3;6.3. Concepts and design of FRET sensors;273
12.4;6.4. Small molecule-based FRET sensors and their applications;280
12.5;6.5. Perspectives and future directions;298
12.6;Acknowledgment;300
12.7;References;300
13;Chapter 7: FilterFRET: Quantitative imaging of sensitized emission;314
13.1;7.1. Introduction;315
13.2;7.2. Two-channel ratio imaging;320
13.3;7.3. Three-channel measurements: FilterFRET;325
13.4;7.4. Optimizing image acquisition;339
13.5;7.5. Postacquisition improvements and analysis;348
13.6;7.6. Discussion;356
13.7;7.A. Appendix;358
13.8;Acknowledgments;372
13.9;References;373
14;Chapter 8: Spectral imaging and its use in the measurement of Foumlrster resonance energy transfer in living cells;376
14.1;8.1. Introduction;377
14.2;8.2. Understanding spectral imaging;378
14.3;8.3. What is the spectral signature of FRET?;396
14.4;Acknowledgments;413
14.5;Appendix;413
14.6;References;416
15;Chapter 9: Total internal reflection fluorescence lifetime imaging microscopy;420
15.1;9.1. Introduction: Total internal reflection theory;421
15.2;9.2. TIRF microscopy history and applications;423
15.3;9.3. Combining TIRF with FLIM;425
15.4;9.4. Results and discussion;430
15.5;9.5. Methods;433
15.6;Acknowledgments;435
15.7;References;435
16;Chapter 10: FRET and FLIM applications in plants;438
16.1;10.1. Introduction;439
16.2;10.2. Multicolor and FRET imaging in plants;439
16.3;10.3. Ideal donor-acceptor pairs for in planta FRET applications;441
16.4;10.4. Quantifying FRET-Methods and Pitfalls;445
16.5;10.5. Protoplasts versus intact tissues: Which cell-type to FRET;452
16.6;10.6. Beyond protein-protein interactions-development and use of FRET-based nanosensors;455
16.7;10.7. Protocols for protoplast transfection and particle bombardment of leaf epidermal cells;457
16.8;10.8. Functional assays and stable plant transformation;459
16.9;10.9. Outlook;460
16.10;Acknowledgment;461
16.11;References;462
17;Chapter 11: Biomedical FRET-FLIM applications;472
17.1;11.1. Introduction;473
17.2;11.2. Rationale;474
17.3;11.3. Methods;475
17.4;11.4. Materials;478
17.5;11.5. Biomedical applications;480
17.6;11.6. Conclusions;491
17.7;References;492
18;Chapter 12: Reflections on FRET imaging: Formalism, probes, and implementation;500
18.1;12.1. Introduction;501
18.2;12.2. The FRET formalism revisited;502
18.3;12.3. The "FRET Calculator";506
18.4;12.4. Novel FRET methods;508
18.5;12.5. FRET probes;511
18.6;12.6. Quo vadis;529
18.7;Acknowledgments;530
18.8;References;530
19;Glossary;544
19.1;Explanation of symbols used in the text;544
20;Index;546
21;Color Plate Section;560