Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 937
fluorescence polarization, 661–663
metal–ligand complexes, 691–693
surface plasmon-coupled emission, 867
time-resolved, 659–660
Immunoglobulin G, 854
Immunoglobulins
anisotropy decay, 396–397
domain-to-domain motions in, 690
fragment, Perrin plot, 371–372
rotational correlation time, 367
Immunophilin KFB59-1, 592–593
Impulse response function, 106–107, 158, 246
Inchworm motion, 784
Incomplete labeling effects, energy-transfer studies, 452, 487–489
Indicators, principles, 16–17
Indo-1, 647, 648, 649, 650
Indole
covalent adduct formation, 314
decay times, 489
electronic state resolution, 360–361
frequency-domain lifetime measurements, 178–180, 185
intensity decays, 579
mixture lifetime resolution, 138–141
quenching, 11, 278, 279, 341, 537
RET in three dimensions, 509–510
solvent effects, 533–534
spectral relaxation, 596
time-domain lifetime, multi-exponential decays, 133, 134
Infrared and NIR fluorescence
dyes, 74–75
light sources, 32
Inner filter effects, 55–56, 290
In situ DNA hybridization, 715–717
Instrumentation, 21–24
corrected emission spectra, 52–54
comparison with known emission spectra, 52–53
conversion between wavelength and wavenumber, 53–54
correction factors obtained with standard lamp, 53
quantum counter and scatterer use, 53
corrected excitation spectra, 51–52
dual-color fluorescence cross correlation spectroscopy, 824
excitation and emission spectra, 21–24, 27–31
distortions in, 30–31
ideal spectrofluorometer, 30
fluorescence correlation spectroscopy, 22–23
frequency-domain lifetime measurements, 163–168, 194–175
cross-correlation detection, 166
frequency synthesizers, 167
history, 163–164
laser diode excitation, 174
LED excitation, 174–175
light modulators, 165–166
200-MHz fluorometers, 164–165
photomultiplier tubes, 167–168
principles of measurement, 168
radio-frequency amplifiers, 167
simple frequency-domain instruments, 173–175
light absorption and deviation from Beer-Lambert law, 58–59
light sources, 31–34
arc and incandescent lamps, 31–34
literature references, 838
monochromators, 34–38
calibration of, 38
polarization characteristics of, 36
second-order transmission in, 37
stray light in, 36–37
wavelength resolution and emission spectra, 35
multiphoton excitation, 21–22
optical filters, 38–41
bandpass, 39
combination, 40
for fluorescence microscopy, 41
neutral density, 40
signal purity and, 41–44
thin-film, 39–40
photomultiplier tubes, 44–49
CCD detectors, 49
failure of, symptoms, 49
hybrid, 49
photon counting vs. analog detection of fluorescence, 48–49
PMT design and dynode chains, 46–47
polarizers, 49–51
spectral response, 45–46
time response of, 47
quantum counters, 51–52
quantum yield standards, 54–55
sample geometry effects, 55–57
sample preparation, common errors in, 57–58
single-molecule detection, 23–24, 764–768
time-correlated single-photon counting (See Time-correlated
single-photon counting (TCSPC))
two-photon and multiphoton excitation, 21–22
Instrument response function (IRF), 100, 105–106, 387, 487
Insulin, 813
Integrins, energy transfer, 518–519
Intensity-based sensing, 645
Intensity decay, 98, 577–578
aromatic amino acids, 578
correlation time imaging, 406–407
least-squares analysis, 161–162
microsecond luminescence decays, 129
multi-exponential model, 101–102
of NADH, 172
RET in three dimensions, 509–511
ribonuclease T I and single-exponential intensity, 584, 585
time-domain lifetime measurements, 145–148
chlorophyll aggregates in hexane, 146–147
FAD, 147–148
green fluorescent protein, systematic data errors, 145–146
picosecond decay time, 146
single-tryptophan protein, 145
tryptophan, 580–583
decay-associated emission spectra, 581
neutral tryptophan derivatives, 581–582
rotamer model, 578–580
tyrosine, 580–583
neutral tyrosine derivatives, 582–583
Intensity decay laws, 141–144
lifetime distributions, 143
multi-exponential decay, 141–143
stretched exponentials, 144
transient effects, 144–145
Intensity fluctuations, 798–799, 800
Interference filters, 39–40
Interferon-γ
resonance energy transfer, 540–542