Cover......Page 1
Half Title......Page 2
Title Page......Page 4
Copyright Page......Page 5
Table of Contents......Page 6
Abbreviations......Page 10
Preface......Page 14
Wavelength, excitation and emission spectra......Page 16
Stokes' shift......Page 20
Quantum yield......Page 21
Relationship between quantum yield, intrinsic lifetime and measured lifetime......Page 22
Fluorophore-solvent interactions......Page 24
Polarization......Page 26
Fluorescence resonance energy transfer (FRET)......Page 27
Advantages of fluorescence microscopy......Page 30
Numerical aperture......Page 31
Resolution......Page 32
Magnification......Page 34
Optical paths of fluorescence microscopes......Page 35
Excitation light sources......Page 38
Wavelength selection devices......Page 40
Objectives......Page 46
Detectors......Page 50
Specimen chambers and stages......Page 52
Arc lamp alignment......Page 54
Optimizing image resolution......Page 56
Photobleaching......Page 57
Cleaning the microscope optics......Page 58
Further tips for optimal fluorescence microscopy......Page 59
Immunofluorescence......Page 62
Fluorescence in situ hybridization (FISH)......Page 65
Fluorescence/differential interference contrast (DIC)......Page 66
Automated fluorescence image cytometry (AFIC)......Page 68
Fluorescence recovery after photobleaching (FRAP)......Page 69
Total internal reflectance fluorescence microscopy (TIRF)......Page 70
Fluorescence resonance energy transfer microscopy (FRETM)......Page 71
Fluorescence lifetime imaging microscopy (FLIM)......Page 74
Delayed luminescence microscopy......Page 76
Green fluorescent protein (GFP)......Page 79
Potential difficulties in using GFP fluorescence......Page 83
Absorbance versus fluorescence......Page 84
Calibration......Page 86
Practical considerations......Page 87
Digital imaging......Page 90
Imaging detectors......Page 91
Image acquisition and digital image processing......Page 96
Ion (ratio) imaging......Page 97
Multiparameter digitized video microscopy (MDVM)......Page 100
Caged compound release/photoactivation......Page 101
Principle of single versus multiphoton microscopy......Page 104
Confocal microscopy......Page 105
Multiphoton microscopy......Page 109
Three-dimensional wide-field microscopy......Page 111
Specimen preparation......Page 114
Optimization of the microscope image......Page 115
Film......Page 116
The darkroom: film development and printing......Page 117
The digital darkroom: presentation of electronically (analogue or digital video) acquired data......Page 118
Appendix A: General optical system problems in fluorescence microscopy......Page 122
Appendix B: General electrical system problems in fluorescence microscopy......Page 124
Appendix C: Poor photographic results and their solutions......Page 126
Appendix D: Difficulties in microfluorometry......Page 130
Appendix E: Absorption and emission maxima for common fluorophores......Page 132
Appendix F: Further reading......Page 138
Appendix G: University/educational WWW sites for microscopy and microanalysis......Page 163
Appendix H: Commercial and non-profit WWW sites for microscopy and microanalysis......Page 169
Appendix I: Glossary......Page 177
Index......Page 182