FLUORESCENCE IN BIOPHYSICS: ACCOMPLISHMENTS AND DEFICIENCIES
CONTENTS 1 THE THEORY OF TWO-PHOTON-INDUCED FLUORESCENCE ANISOTROPY 1 1 1 2 1 3 1 4 1 5 1 6 INTRODUCTION TWO-PHOTON ABSORPTION 1 2 1 1 2 2 1 2 3 1 3 1 1 3 2 1 3 3 1 3 4 1 3 5 1 4 1 1 4 2 1 4 3 1 4 4 1 5 1 1 5 2 1 5 3 1 5 4 1 5 5 QUALITATIVE ASPECTS A BRIEF HISTORY QUANTITATIVE DESCRIPTION ANISOTROPY THEORY INTRODUCTION AND SCOPE EXCITATION WITH LINEARLY POLARIZED LIGHT EXCITATION WITH CIRCULARLY POLARIZED LIGHT THE TWO-DIMENSIONAL LIMIT WITH LINEAR POLARIZATION THREE-DIMENSIONAL TENSORS COMPARISON WITH EXPERIMENT INTRODUCTION POLYENES BENZENE DERIVATIVES INDOLES SOME PREDICTIONS PHENYLALANINE TYROSINE INDOLE IN NONPOLAR SOLVENT 5-METHOXYINDOLE NUCLEOTIDES CONCLUDING REMARKS REFERENCES 2 ANISOTROPY DECAYS INDUCED BY TWO-PHOTON EXCITATION CAREY K JOHNSON AND CHAOZHI WAN 2 1 INTRODUCTION 2 1 1 THEORETICAL BACKGROUND 2 1 2 TIME-RESOLVED TWO-PHOTON EXCITATION 2 2 ORIENTATIONAL DISTRIBUTIONS INDUCED BY TWO-PHOTON EXCITATION 2 2 1 ORIENTATIONAL AVERAGES AND SPHERICAL TENSORS 2 2 2 SPHERICAL TENSORS: A PRIMER 2 2 3 TWO-PHOTON PHOTOSELECTION 2 2 4 TWO-PHOTON ANISOTROPIES 2 2 5 TWO-PHOTON ANISOTROPIC DISTRIBUTIONS 2 2 6 THE SPECIAL CASE OF A TENSOR 2 3 TWO-PHOTON ANISOTROPY DECAY 2 3 1 ROTATIONAL DIFFUSION 2 3 2 TWO-PHOTON-INDUCED REORIENTATIONAL DECAY 2 3 3 TIME DEPENDENCE OF THE TWO-PHOTON ANISOTROPIES 2 3 4 EXAMPLE: PERYLENE 2 4 CONCLUSION REFERENCES 3 MULTIPHOTON EXCITATION OF BIOCHEMICAL FLUOROPHORES JOSEPH R LAKOWICZ AND IGNACY GRYCZYNSKI 3 1 INTRODUCTION 3 2 TWO-PHOTON EXCITATION OF DPH 3 2 1 EMISSION SPECTRA AND DPH INTENSITY DECAYS FOR 1PE AND 2PE 3 2 2 ANISOTROPY SPECTRA WITH 2PE 3 2 3 THEORY OF PHOTOSELECTION WITH MPE 3 2 4 ANISOTROPY DECAYS OF DPH WITH 2PE 3 2 5 TWO-PHOTON EXCITATION OF DPH IN MEMBRANES 3 3 TWO-PHOTON EXCITATION OF FLUOROPHORE-STAINED DNA 3 3 1 EMISSION SPECTRA OF HOECHST 33342 3 3 2 FLUORESCENCE INTENSITY DECAYS OF HOECHST 33342 3 3 3 ANISOTROPY EXCITATION SPECTRA OF HOECHST IN GLYCEROL AT –20°C 3 3 4 ANISOTROPY EXCITATION SPECTRA OF HOECHST 33342-DNA COMPLEX 3 4 TWO-PHOTON EXCITATION OF INDOLE, TRYPTOPHAN, AND PROTEINS 3 4 1 EMISSION SPECTRA 3 4 2 ANISOTROPY SPECTRA 3 4 3 TWO-PHOTON EXCITATION OF HSA 3 5 TWO-PHOTON-INDUCED FLUORESCENCE OF ALKANES 3 6 STANDARDS FOR 2PE 3 6 1 A PS TWO-PHOTON STANDARD 3 7 THREE-PHOTON EXCITATION OF FLUORESCENCE 3 7 1 THREE-PHOTON EXCITATION OF 2,5-DIPHENYLOXAZOLE 3 7 2 THREE-PHOTON EXCITATION OF DPH IN MEMBRANES 3 7 3 THREE-PHOTON EXCITATION OF THE CALCIUM PROBE INDO-1 3 7 4 THREE-PHOTON EXCITATION OF TRYPTOPHAN AND PROTEINS 3 7 5 PERSPECTIVES ON 3PE 3 8 TWO-COLOR TWO-PHOTON EXCITATION 3 9 FUTURE APPLICATIONS OF MPE REFERENCES 4 TWO-PHOTON EXCITATION AND ANISOTROPY DECAYS IN MEMBRANES AND ORIENTED SYSTEMS B WIEB VAN DER MEER AND S -Y SIMON CHEN 4 1 INTRODUCTION 4 2 TWO-PHOTON ANISOTROPY THEORY IN ISOTROPIC SUSPENSIONS 4 3 TWO-PHOTON ANISOTROPY THEORY IN ORIENTED MEMBRANES 4 4 CONCLUSIONS 4 5 APPENDIX A APPENDIX A 1 APPENDIX A 2 4 6 APPENDIX B APPENDIX B 1 APPENDIX B 2 APPENDIX B 3 4 7 APPENDIX C 4 8 APPENDIX D 4 9 APPENDIX E 4 10 APPENDIX F 4 11 APPENDIX G 4 12 APPPENDIX H REFERENCES 5 TWO-PHOTON INDUCED FLUORESCENCE OF PROTEINS BORYS KIERDASZUK, IGNACY GRYCZYNSKI, AND JOSEPH R LAKOWICZ 5 1 INTRODUCTION 5 2 EXCITATION AND EMISSION SPECTRA OF AROMATIC AMINO ACIDS FOR ONE- AND TWO-PHOTON EXCITATION 5 2 1 STUDIES OF TRYPTOPHAN, TYROSINE, AND PHENYLALANINE IN SOLUTION 5 2 2 TRYPTOPHAN AND TYROSINE IN PROTEINS 5 3 FLUORESCENCE ANISOTROPY OF ONE- AND TWO-PHOTON EXCITATION OF TRYPTOPHAN AND TYROSINE 5 3 1 LIMITING-ANISOTROPY SPECTRA 5 3 2 ANISOTROPY OF TRYPTOPHAN- AND/OR TYROSINE-CONTAINING PROTEINS 5 4 TWO-PHOTON INDUCED FLUORESCENCE OF PROTEIN-BOUND CHROMOPHORES 5 5 EXPERIMENTAL METHODS REFERENCES 6 FLUORESCENCE AND MULTIWAVE MIXING INDUCED BY PHOTON ABSORPTION OF EXCITED MOLECULES VALERY L BOGDANOV 6 1 INTRODUCTION 6 2 EMISSION OF HIGHLY EXCITED MOLECULES 6 2 1 METHODS OF EXCITATION 6 2 2 RADIATIONLESS DECAY OF HIGHLY EXCITED STATES 6 2 3 EMISSION SPECTRA AND RELAXATIONS OF SHORT-LIVED HIGHLY EXCITED STATES 6 2 4 QUANTUM YIELD OF FLUORESCENCE FROM HIGHLY EXCITED STATES 6 2 5 ANISOTROPY OF FLUORESCENCE FROM HIGHLY EXCITED STATES 6 2 6 VIBRATIONAL RELAXATION AND FLUORESCENCE SPECTRA OF HIGHLY EXCITED MOLECULES 6 2 7 ENERGY TRANSFER FROM HIGHLY EXCITED STATES 6 2 8 STEPPED EXCITATION OF BIFLUROPHORE 6 3 TRANSIENT THIRD-ORDER NONLINEARITY AND RELAXATION OF EXCITED MOLECULES 6 3 1 PRELIMINARY REMARKS 6 3 2 DISPERSION OF TRANSIENT SUSCEPTIBILITY AND ULTRAFAST RELAXATION OF HIGHLY EXCITED MOLECULES 6 3 3 TRANSIENT NONLINEAR SUSCEPTIBILITY AND ORIENTATIONAL ANISOTROPY OF EXCITED MOLECULES 6 3 4 TIME-RESOLVED DISPERSION OF TRANSIENT SUSCEPTIBILITY AND SPECTRAL DYNAMICS OF EXCITED MOLECULES 6 4 SUMMARY REFERENCES 7 ULTRAFAST STIMULATED-EMISSION SPECTROSCOPY G J BLANCHARD 7 1 STIMULATED-EMISSION PUMPING SPECTROSCOPIES 7 2 EXPERIMENTAL IMPLEMENTATION OF ULTRAFAST STIMULATED-EMISSION PUMP–PROBE SPECTROSCOPY 7 3 APPLICATIONS OF STIMULATED-EMISSION SPECTROSCOPY TO ROTATIONAL DIFFUSION DYNAMICS MEASUREMENTS 7 4 MEASURING VIBRATIONAL POPULATION RELAXATION USING STIMULATED EMISSION 7 4 1 CHEMICAL INFORMATION CONTENT OF ULTRAFAST STIMULATED-EMISSION MEASUREMENTS: PERYLENE AND 1-METHYLPERYLENE IN N-ALKANES 7 4 2 RELAXATION IN DIFFERENT ELECTRONIC STATES 7 4 3 SOLVENT-DEPENDENT RELAXATION DYNAMICS OF PERYLENE 7 4 4 UNDERSTANDING THE LENGTH SCALES OVER WHICH INTERMOLECULAR VIBRATIONAL ENERGY TRANSFER OPERATES 7 4 5 1-METHYLPERYLENE VIBRATIONAL POPULATION RELAXATION 7 4 6 REORIENTATION DYNAMICS OF 1-METHYLPERYLENE IN THE ALKANES 7 4 7 COMPARISON OF AND DATA FOR 1-METHYLPERYLENE 7 5 SUMMARY AND OVERVIEW REFERENCES 8 FLUORESCENCE QUENCHING BY STIMULATED EMISSION JOSEPH R LAKOWICZ AND IGNACY GRYCZYNSKI 8 1 INTRODUCTION 8 2 ONE-PULSE AND TWO-PULSE LIGHT QUENCHING 8 2 1 ONE-PULSE TIME-COINCIDENT LIGHT QUENCHING 8 2 2 TWO-PULSE TIME-DELAYED LIGHT QUENCHING 8 3 EFFECTS OF LIGHT QUENCHING ON TIME-RESOLVED INTENSITY AND ANISOTROPY DECAYS 8 4 LIFETIME-RESOLVED EXPERIMENTS BY TWO-BEAM LIGHT QUENCHING 8 5 EXPERIMENTAL RESULTS ON ONE-PULSE LIGHT QUENCHING 8 5 1 ONE-PULSE LIGHT QUENCHING OF DCM 8 5 2 ONE-PULSE LIGHT QUENCHING WITH TWO-PHOTON EXCITATION 8 6 EXPERIMENTAL RESULTS ON TWO-PULSE LIGHT QUENCHING 8 6 1 STEADY-STATE MEASUREMENTS WITH TWO-BEAM LIGHT QUENCHING 8 6 2 TIME-DOMAIN AND FREQUENCY-DOMAIN STUDIES OF LIGHT QUENCHING 8 6 3 WAVELENGTH-SELECTIVE LIGHT QUENCHING WITH TIME-DEPENDENT SPECTRAL RELAXATION 8 7 APPLICATIONS OF LIGHT QUENCHING 8 7 1 WAVELENGTH- OR LIFETIME-SELECTIVE LIGHT QUENCHING 8 7 2 LIGHT QUENCHING WITH TOTAL INTERNAL REFLECTANCE 8 8 DISCUSSION 8 8 1 COMPARISON OF LIGHT QUENCHING AND INDUCED STIMULATED EMISSION 8 8 2 BIOPHYSICAL APPLICATIONS OF LIGHT QUENCHING 8 8 3 PULSE-WIDTH-DEPENDENT LIGHT QUENCHING 8 8 4 PERSPECTIVES ON LIGHT QUENCHING REFERENCES 9 INCREASING THE RESOLUTION OF FAR-FIELD FLUORESCENCE LIGHT MICROSCOPY BY POINT-SPREAD-FUNCTION ENGINEERING STEFAN W HELL 9 1 INTRODUCTION 9 1 1 WHY FAR-FIELD LIGHT MICROSCOPY? 9 1 2 IMAGING WITH A LENS: THE POINT-SPREAD FUNCTION 9 1 3 SCANNING FLUORESCENCE MICROSCOPY 9 2 PSF ENGINEERING THROUGH FLUORESCENCE INHIBITION 9 2 1 STED FLUORESCENCE MICROSCOPY 9 2 2 GROUND-STATE-DEPLETION FLUORESCENCE MICROSCOPY 9 3 PSF ENGINEERING THROUGH APERTURE INCREASE: 4PI CONFOCAL MICROSCOPY 9 3 1 4PI ILLUMINATION AND DETECTION PSF 9 3 2 THE 4PI CONFOCAL MICROSCOPE AND ITS IMAGING MODES 9 3 3 TWO-PHOTON EXCITATION 4PI MICROSCOPY 9 3 4 CONFOCAL TWO-PHOTON EXCITATION 4PI MICROSCOPY 9 3 5 LIMITATIONS AND FURTHER POTENTIALS OF 4PI CONFOCAL MICROSCOPY 9 4 OTHER EXAMPLES OF PSF ENGINEERING 9 4 1 OFFSET-BEAM OVERLAP MICROSCOPY 9 4 2 THETA-4PI CONFOCAL MICROSCOPY 9 5 RECENT DEVELOPMENTS 9 5 1 STED 4PI CONFOCAL MICROSCOPY 9 5 2 4PI CONFOCAL MICROSCOPY COMBINED WITH IMAGE RESTORATION PROVIDES 3D-RESOLUTION IN THE 100 NM RANGE 9 6 CONCLUSION REFERENCES 10 TIME-RESOLVED STIMULATED-EMISSION AND TRANSIENT-ABSORPTION MICROSCOPY AND SPECTROSCOPY P T C SO, C Y DONG, K M BERLAND, T FRENCH, AND E GRATTON 10 1 INTRODUCTION 10 1 1 PUMP–PROBE SPECTROSCOPY 10 1 2 TIME-RESOLVED FLUORESCENCE MICROSCOPY 10 1 3 OVERVIEW 10 2 THEORY 10 2 1 PUMP–PROBE SPECTROSCOPY 10 2 2 PUMP–PROBE MICROSCOPY 10 3 INSTRUMENTATION AND METHODS 10 3 1 PUMP–PROBE LIGHT SOURCES 10 3 2 OPTICS FOR ABSORPTION-MODE MEASUREMENT 10 3 3 OPTICS FOR STIMULATED-EMISSION MODE 10 3 4 SCANNER OPTICS FOR MICROSCOPY 10 3 5 SIGNAL DETECTION AND PROCESSING 10 4 SPECTROSCOPY APPLICATIONS AND PERFORMANCE 10 4 1 PUMP–PROBE ABSORPTION SPECTROSCOPY 10 4 2 STIMULATED-EMISSION PUMP–PROBE SPECTROSCOPY 10 5 APPLICATIONS AND PERFORMANCES OF PUMP–PROBE FLUORESCENCE MICROSCOPE 10 5 1 LINEARITY OF PUMP–PROBE SIGNAL RESPONSE ON LASER POWER LEVEL 10 5 2 EXPERIMENTAL VERIFICATION OF PUMP–PROBE MICROSCOPY RESOLUTION 10 5 3 TIME-RESOLUTION OF PUMP–PROBE MICROSCOPY 10 5 4 COMPARISON BETWEEN CONVENTIONAL MICROSCOPY AND PUMP–PROBE FLUORESCENCE MICROSCOPY IN HUMAN ERYTHROCYTES AND MOUSE FIBROBLASTS 10 5 5 MULTIPLE-DYE-LABELING APPLICATION 10 6 CONCLUSION REFERENCES 11 MULTIPHOTON EXCITATION OF MOLECULAR FLUOROPHORES AND NONLINEAR LASER MICROSCOPY CHRIS XU AND WATT W WEBB 11 1 INTRODUCTION 11 2 MULTIPHOTON SPECTROSCOPY 11 2 1 ESTIMATIONS OF MULTIPHOTON CROSS SECTION 11 2 2 THEORETICAL ANALYSIS OF MPE 11 2 3 TWO- AND THREE-PHOTON EXCITATION SPECTRA 11 2 4 POWER DEPENDENCE OF TWO- AND THREE-PHOTON-EXCITED FLUORESCENCE 11 2 5 DEPENDENCE OF TWO-PHOTON-EXCITED FLUORESCENCE ON PULSE WIDTH 11 2 6 ONE-, TWO-, AND THREE-PHOTON-EXCITED FLUORESCENCE EMISSION SPECTRA COMPARED 11 2 7 TWO-PHOTON SPECTRA EXCITED BY CW AND FEMTOSECOND PULSED LASER COMPARED 11 3 MULTIPHOTON MICROSCOPY 11 3 1 INSTRUMENT OF NONLINEAR MICROSCOPY 11 3 2 MULTIPHOTON EXCITATION VOLUME 11 3 3 MULTIPHOTON UNCAGING 11 3 4 IMAGING OF INTRINSIC FLUOROPHORES WITH MULTIPHOTON EXCITATION 11 3 5 DEEP IMAGING 11 4 CONSIDERATIONS IN MPE SPECTROSCOPY AND MICROSCOPY 11 4 1 FLUOROPHORE GROUND-STATE DEPLETION 11 4 2 INTERSYSTEM CROSSING AND PHOTOBLEACHING 11 4 3 LASER-MATTER INTERACTION AT HIGH INTENSITY THE THEORY OF TWO-PHOTON-INDUCED FLUORESCENCE ANISOTROPY 1 1 INTRODUCTION FLUORESCENCE ANISOTROPY (PHOTOSELECTION) TECHNIQUES HAVE PLAYED AN IMPOR- TANT ROLE IN ELUCIDATING THE NATURE OF EXCITED ELECTRONIC STATES IN POLYATOMIC MOLECULES AND THEIR ROTATIONAL DYNAMICS FOR MANY YEARS (1–3) IN THE CLASSIC EXPERI- MENTS SINGLE PHOTONS OF LINEARLY POLARIZED LIGHT ARE ABSORBED BY A RANDOMLY ORIENTED SAMPLE, CREATING A PARTIALLY ORIENTED (PHOTOSELECTED) ENSEMBLE OF EXCITED MOLECULES IN THE USUAL ELECTRIC DIPOLE MECHANISM THE PROBABILITY OF ABSORPTION GOES AS THE SQUARE OF THE PROJECTION OFTHE MOLECULE’S TRANSITION DIPOLE VECTOR ONTO THE POLARIZATION DIRECTION OF THE LIGHT THE AVERAGE ORIENTATION IS REVEALED BY THE POLARIZATION (ANISOTROPY) OF THE SUBSEQUENTLY EMITTED PHOTONS, PROVIDED THERE IS LITTLE MOLECULAR ROTATION PRIOR TO EMISSION OF THE SECOND PHOTON THE RATE OF LOSS OF THE ANISOTROPY IS DIRECTLY RELATED TO THE ROTATIONAL DIFFUSION RATE DURING THE EXCITED- STATE LIFETIME THE WIDESPREAD AVAILABILITY OF HIGH REP-RATE PICOSECOND PULSED LASERS HAS MADE TWO-PHOTON EXCITATION (TPE) MORE ACCESSIBLE IN RECENT YEARS, AND WITH IT A DESIRE TO EXPLOIT THE FLUORESCENCE ANISOTROPY CREATED FROM TPE (4– 17) HOWEVER, IT IS FAIR TO SAY THAT MOST SPECTROSCOPISTS TODAY ARE NOT VERY FAMILIAR WITH THE TWO- PHOTON ABSORPTION PROCESS IT IS THE PURPOSE OF THIS CHAPTER TO PRESENT A FORMAL THEORY OF STEADY-STATE ANISOTROPY OF FLUORESCENCE PRODUCED BY TPE AND TO REVIEW SOME OF THE MAJOR DIFFERENCES BETWEEN ONE- AND TWO-PHOTON SPECTROSCOPY AND HOW THEY TRANSLATE INTO DIFFERENCES IN THE ANISOTROPY OF THE FLUORESCENCE THE SCOPE OF THIS CHAPTER IS LIMITED TO A SUBSPACE OF THE ELEGANT TREATMENT OF GENERAL THREE-PHOTON PROCESSES BY MCCLAIN,(18,19) WHEREIN ONLY THE CASE OF TWO IDENTICAL PHOTONS ABSORBED (ONE-COLOR CASE) FOLLOWED BY FLUORESCENCE (AT