Fluorescence Microscopy

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