Principles of fluorescence spectroscopy:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
New York, NY
Springer
2010
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| Ausgabe: | 3. ed. (corr. at 4. print.) |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Hier auch später erschienene, unveränderte Nachdrucke |
| Beschreibung: | XXVI, 954 S. Ill., zahlr. graph. Darst. |
| ISBN: | 0387312781 9780387312781 9781489978806 |
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| 020 | |a 9780387312781 |c hbk. |9 978-0-387-31278-1 | ||
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| 100 | 1 | |a Lakowicz, Joseph R. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Principles of fluorescence spectroscopy |c Joseph R. Lakowicz |
| 250 | |a 3. ed. (corr. at 4. print.) | ||
| 264 | 1 | |a New York, NY |b Springer |c 2010 | |
| 300 | |a XXVI, 954 S. |b Ill., zahlr. graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Hier auch später erschienene, unveränderte Nachdrucke | ||
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Datensatz im Suchindex
| _version_ | 1804143570325602304 |
|---|---|
| adam_text | Contents
I. Introduction to Fluorescence
1.1. Phenomena of Fluorescence
.....................................
I
1.2.
Jabłoński
Diagram
.................................................... 3
1.3.
Characteristics of Fluorescence Ivniission
................ 6
1
.3.
1
.
The Stokes Shift
............................................ 6
1.3.2.
Emission Spectra Are Typically Inilcpenilent
of the Excitation Wavelength
........................ 7
1
.3.3.
Exceptions
to the Mirror-Image Rule
...........
X
1.4.
Fluorescence Lifetimes anil Quantum Yields
........... 9
1.4.1.
Fluorescence Quenching
...............................
I
1
1
.4.2.
Timescale of Molecular Processes
in Solution
..................................................... 12
1
.5.
Fluorescence Anisotropy
..........................................
1
2
1.6.
Resonance Energy Transfer
...................................... 13
1.7.
Steady-Slate and Time-Resolved Fluorescence
....... 14
1.7.1.
Why Time-Resolved Measurements?
............ 15
1.8.
Biochemical Fluorophores
....................................... 15
1.8.1.
Fluorescent Indicators
................................... 16
1.9.
Molecular Information from Fluorescence
.............. 17
1
.9.
1
.
Hmission Spectra and the Stokes Shift
......... 17
1.9.2.
Quenching of Fluorescence
........................... 18
1.9.3.
Fluorescence Polarization or Anisotropy
...... 19
1.9.4.
Resonance Energy Transfer
........................... 19
1.
10.
Biochemical Examples of Basic Phenomena
........... 20
1.11.
New Fluorescence Technologies
.............................. 21
1.11.1.
Multiphoton Excitation
............................... 2
1
1.11.2.
Fluorescence Correlation Spectroscopy
...... 22
I.I
1.3.
Single-Molecule Detection
.......................... 23
1.12.
Overview of Fluorescence Spectroscopy
................. 24
References
................................................................ 25
Problems
................................................................... 25
2.
Instrumentation for Fluorescence
Spectroscopy
2.1.
Spectrofluorometers
................................................... 27
2.1.1.
Spectrofluorometers for Spectroscopy
Research
........................................................ 27
2.
1
.2.
Spectrofluorometers for High Throughput
... 29
2.1.3.
An Ideal Spectrofluorometer
......................... 30
2.1.4.
Distortions in Excitation and Emission
Spectra
........................................................... 30
2.2.
Light Sources
........................................................... 31
2.2.1.
Arc Lamps and Incandescent
Xenon Lamps
................................................ 31
2.2.2.
Pulsed Xenon Lamps
.................................... 32
2.2.3.
High-Pressure Mercury (Hg) Lamps
............ 33
2.2.4.
Xe Hg Arc Lamps
........................................ 33
2.2.5.
Quart/Tungsten Halogen (QTH) Lamps
..... 33
2.2.6.
Low-Pressure Hg and
Hg Ar
Lamps
............ 33
2.2.7.
LED Light Sources
.........................................13
2.2.8.
Laser Diodes
.................................................. 34
2.3.
Monochromators
...................................................... 34
2.3.1.
Wavelength Resolution and Emission
Spectra
........................................................... 35
2.3.2.
Polarization Characteristics of
Monochromators
........................................... 36
2.3.3.
Stray Light in Monochromators
.................... 36
2.3.4.
Second-Order Transmission in
Monochromators
........................................... 37
2.3.5.
Calibration of Monochromators
.................... 38
2.4.
Optical Filters
........................................................... 38
2.4.1.
Colored Filters
............................................... 38
2.4.2.
Thin-Film Filters
........................................... 39
2.4.3.
Filter Combinations
....................................... 40
2.4.4.
Neutral-Density Filters
.................................. 40
2.4.5.
Filters for Fluorescence Microscopy
............. 41
2.5.
Optical Filters and Signal Purity
.............................. 41
2.5.1.
Emission Spectra Taken through Filters
....... 43
2.6.
Photomultiplier Tubes
.............................................. 44
2.6.1.
Spectral Response of PMTs
.......................... 45
2.6.2.
PMT Designs and Dynode Chains
................ 46
2.6.3.
Time Response of Photomultiplier Tubes
..... 47
2.6.4.
Photon Counting versus Analog Detection
of Fluorescence
............................................. 48
2.6.5.
Symptoms of PMT Failure
............................ 49
2.6.6.
CCD
Detectors
.............................................. 49
2.7.
Polarizers
.................................................................. 49
2.8.
Corrected Excitation Spectra
.................................... 51
2.8.
1
.
Corrected Excitation Spectra Using
a Quantum Counter
....................................... 51
2.9.
Corrected Emission Spectra
..................................... 52
2.9.
1
.
Comparison with Known Emission
Spectra
........................................................... 52
2.9.2.
Corrections Using a Standard Lamp
............. 53
2.9.3.
Correction Factors Using a Quantum
Counter and Scatterer
.................................... 53
CONTENTS
2.9.4.
Conversion
between Wavelength and
Wavenumber
.................................................. 53
2.10.
Quantum Yield Standards
......................................... 54
2.11.
Effects of Sample Geometry
.................................... 55
2.12.
Common Errors in Sample Preparation
................... 57
2.13.
Absorption of Light and Deviation from the
Beer-Lambert Law
.................................................... 58
2.13.1.
Deviations from Beer s Law
........................ 59
2.14.
Conclusions
.............................................................. 59
References
................................................................ 59
Problems
................................................................... 60
3.
Fluorophores
3.1.
Intrinsic or Natural Fluorophores
............................. 63
3.1.1.
Fluorescence Enzyme Cofactors
................... 63
3.1.2.
Binding of NADH to a Protein
..................... 65
3.2.
Extrinsic Fluorophores
............................................. 67
3.2.1.
Protein-Labeling Reagents
............................ 67
3.2.2.
Role of the Stokes Shift in Protein
Labeling
......................................................... 69
3.2.3.
Photostability of Fluorophores
...................... 70
3.2.4.
Non-Covalent Protein-Labeling
Probes
............................................................ 71
3.2.5.
Membrane Probes
.......................................... 72
3.2.6.
Membrane Potential Probes
.......................... 72
3.3.
Red and Near-Infrared (NIR) Dyes
.......................... 74
3.4. DNA
Probes
............................................................. 75
3.4.1. DNA
Base Analogues
................................... 75
3.5.
Chemical Sensing Probes
......................................... 78
3.6.
Special Probes
.......................................................... 79
3.6.1.
Fluorogenic Probes
........................................ 79
3.6.2.
Structural Analogues of Biomolecules
.......... 80
3.6.3.
Viscosity Probes
............................................ 80
3.7.
Green Fluorescent Proteins
...................................... 81
3.8.
Other Fluorescent Proteins
....................................... 83
3.8.1.
Phytofluors: A New Class of
Fluorescent Probes
........................................ 83
3.8.2.
Phycobiliproteins
........................................... 84
3.8.3.
Specific Labeling of Intracellular
Proteins
.......................................................... 86
3.9.
Long-Lifetime Probes
.............................................. 86
3.9.1. Lanthanides................................................... 87
3.9.2.
Transition Metal-Ligand Complexes
............ 88
3.10.
Proteins as Sensors
................................................... 88
3.11.
Conclusion
................................................................ 89
References
................................................................ 90
Problems
................................................................... 94
4.
Time-Domain Lifetime Measurements
4.1.
Overview of Time-Domain and Frequency-
Domain Measurements
............................................. 98
4.1.1.
Meaning of the Lifetime or Decay Time
...... 99
4.1.2.
Phase and Modulation Lifetimes
.................. 99
4.1.3.
Examples of Time-Domain and
Frequency-Domain Lifetimes
....................... 100
4.2. Biopolymers
Display Multi-Exponential or
Heterogeneous Decays
............................................. 101
4.2.1.
Resolution of Multi-Exponential
Decays Is Difficult
........................................ 103
4.3.
Time-Correlated Single-Photon Counting
............... 103
4.3.1.
Principles of TCSPC
..................................... 104
4.3.2.
Example of TCSPC Data
.............................. 105
4.3.3.
Convolution Integral
...................................... 106
4.4.
Light Sources for TCSPC
........................................ 107
4.4.1.
Laser Diodes and Light-Emitting Diodes
..... 107
4.4.2.
Femtosecond Titanium Sapphire Lasers
....... 108
4.4.3.
Picosecond Dye Lasers
................................. 110
4ЛЛ.
Flashlamps
..................................................... 112
4.4.5.
Synchrotron Radiation
.................................. 114
4.5.
Electronics for TCSPC
............................................. 114
4.5.1.
Constant Fraction Discriminators
................. 114
4.5.2.
Amplifiers
...................................................... 115
4.5.3.
Time-to-Amplitude Converter
(TAC)
and Analyte-to-Digital Converter (ADC)
...... 115
4.5.4.
Multichannel Analyzer
.................................. 116
4.5.5.
Delay Lines
................................................... 116
4.5.6.
Pulse Pile-Up
................................................. 116
4.6.
Detectors for TCSPC
................................................ 117
4.6.1.
MicroChannel Plate PMTs
............................. 117
4.6.2.
Dynode Chain PMTs
..................................... 118
4.6.3.
Compact PMTs
.............................................. 118
4.6.4.
Photodiodes
as Detectors
.............................. 118
4.6.5.
Color Effects in Detectors
............................. 119
4.6.6.
Timing Effects of Monochromators
.............. 121
4.7.
Multi-Detector and Multidimensional TCSPC
........ 121
4.7.1.
Multidimensional TCSPC and
DNA
Sequencing
........................................... 123
4.7.2.
Dead Times, Repetition Rates, and
Photon Counting Rates
.................................. 124
4.8.
Alternative Methods for Time-Resolved
Measurements
........................................................... 124
4.8.1.
Transient Recording
...................................... 124
4.8.2.
Streak Cameras
.............................................. 125
4.8.3.
Upconversion Methods
.................................. 128
4.8.4.
Microsecond Luminescence Decays
............. 129
4.9.
Data Analysis: Nonlinear Least Squares
.................. 129
4.9.1.
Assumptions of Nonlinear Least Squares
..... 130
4.9.2.
Overview of Least-Squares Analysis
............ 130
4.9.3.
Meaning of the Goodness-of-Fit
................... 131
4.9.4.
Autocorrelation Function
.............................. 132
4.10.
Analysis of Multi-Exponential Decays
.................... 133
4.10.1.
p-Terphenyl and
Indole:
Two Widely
Spaced Lifetimes
......................................... 133
4.10.2.
Comparison of
χκ2
Values:
F
Statistic
........ 133
4.10.3.
Parameter Uncertainty: Confidence
Intervals
....................................................... 134
4.10.4.
Effect of the Number of Photon Counts
..... 135
4.10.5.
Anthranilic Acid and 2-Aminopurine:
Two Closely Spaced Lifetimes
.................... 137
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
xvii
4.10.6.
Global Analysis: Multi-Wavelength
Measurements
.............................................. 138
4.10.7.
Resolution of Three Closely Spaced
Lifetimes
...................................................... 138
4.11.
Intensity Decay Laws
............................................... 141
4.11.1.
Multi-Exponential Decays
.......................... 141
4.11.2.
Lifetime Distributions
................................. 143
4.11.3.
Stretched Exponentials
................................ 144
4.11.4.
Transient Effects
.......................................... 144
4.12.
Global Analysis
........................................................ 144
4.13.
Applications of TCSPC
............................................ 145
4.13.1.
Intensity Decay for a Single Tryptophan
Protein
......................................................... 145
4.13.2.
Green Fluorescent Protein: Systematic
Errors in the Data
........................................ 145
4.13.3.
Picosecond Decay Time
.............................. 146
4.13.4.
Chlorophyll Aggregates in
Hexane
............. 146
4.13.5.
Intensity Decay of Flavin
Adeninę
Dinucleotide (FAD)
..................................... 147
4.14.
Data Analysis: Maximum Entropy Method
............. 148
References
................................................................ 149
Problems
................................................................... 154
5.
Frequency-Domain Lifetime
Measurements
5.1.
Theory of Frequency-Domain Fluorometry
............. 158
5.1.1.
Least-Squares Analysis of Frequency-
Domain Intensity Decays
.............................. 161
5.1.2.
Global Analysis of Frequency-Domain
Data
............................................................... 162
5.2.
Frequency-Domain Instrumentation
........................ 163
5.2.1.
History of Phase-Modulation
Fluorometers
.................................................. 163
5.2.2.
An MHz Frequency-Domain
Fluorometer....
164
5.2.3.
Light Modulators
........................................... 165
5.2.4.
Cross-Correlation Detection
.......................... 166
5.2.5.
Frequency Synthesizers
................................. 167
5.2.6.
Radio Frequency Amplifiers
......................... 167
5.2.7.
Photomultiplier Tubes
................................... 167
5.2.8.
Frequency-Domain Measurements
............... 168
5.3.
Color Effects and Background Fluorescence
........... 168
5.3.1.
Color Effects in Frequency-Domain
Measurements
................................................ 168
5.3.2.
Background Correction in Frequency-
Domain Measurements
.................................. 169
5.4.
Representative Frequency-Domain Intensity
Decays
...................................................................... 170
5.4.1.
Exponential Decays
....................................... 170
5.4.2.
Multi-Exponential Decays of
Staphylococcal Nuclease and Melittin
.......... 171
5.4.3.
Green Fluorescent Protein: One- and
Two-Photon Excitation
.................................. 171
5.4.4.
SPQ: Collisional Quenching of a
Chloride Sensor
............................................. 171
5.4.5.
Intensity Decay of NADH
............................. 172
5.4.6.
Effect of Scattered Light
............................... 172
5.5.
Simple Frequency-Domain Instruments
.................. 173
5.5.1.
Laser Diode Excitation
.................................. 174
5.5.2.
LED Excitation
.............................................. 174
5.6.
Gigahertz Frequency-Domain Fluorometry
............. 175
5.6.1.
Gigahertz FD Measurements
........................ 177
5.6.2.
Biochemical Examples of Gigahertz
FDData
......................................................... 177
5.7.
Analysis of Frequency-Domain Data
....................... 178
5.7.1.
Resolution of Two Widely Spaced
Lifetimes
........................................................ 178
5.7.2.
Resolution of Two Closely Spaced
Lifetimes
........................................................ 180
5.7.3.
Global Analysis of a Two-Component
Mixture
.......................................................... 182
5.7.4.
Analysis of a Three-Component Mixture:
Limits of Resolution
...................................... 183
5.7.5.
Resolution of a Three-Component
Mixture with a Tenfold Range of
Decay Times
.................................................. 185
5.7.6.
Maximum Entropy Analysis of FD Data
...... 185
5.8.
Biochemical Examples of Frequency-Domain
Intensity Decays
....................................................... 186
5.8.1. DNA
Labeled with DAPI
.............................. 186
5.8.2.
Mag-Quin-2: A Lifetime-Based Sensor
for Magnesium
.............................................. 187
5.8.3.
Recovery of Lifetime Distributions from
Frequency-Domain Data
............................... 188
5.8.4.
Cross-Fitting of Models: Lifetime
Distributions of Melittin
................................ 188
5.8.5.
Frequency-Domain Fluorescence
Microscopy with an LED Light Source
........ 189
5.9.
Phase-Angle and Modulation Spectra
...................... 189
5.10.
Apparent Phase and Modulation Lifetimes
.............. 191
5.11.
Derivation of the Equations for Phase-
Modulation Fluorescence
......................................... 192
5.11.1.
Relationship of the Lifetime to the
Phase Angle and Modulation
...................... 192
5.11.2.
Cross-Correlation Detection
........................ 194
5.12.
Phase-Sensitive Emission Spectra
............................ 194
5.12.1.
Theory of Phase-Sensitive Detection
of Fluorescence
........................................... 195
5.12.2.
Examples of PSDF and Phase
Suppression
................................................. 196
5.12.3.
High-Frequency or Low-Frequency
Phase-Sensitive Detection
........................... 197
5.13.
Phase-Modulation Resolution of Emission
Spectra
...................................................................... 197
5.13.1.
Resolution Based on Phase or Modulation
Lifetimes
...................................................... 198
5.13.2.
Resolution Based on Phase Angles
and Modulations
.......................................... 198
5.13.3.
Resolution of Emission Spectra from
Phase and Modulation Spectra
.................... 198
References
................................................................ 199
Problems
................................................................... 203
xviii
CONTENTS
6. Solvent
and Environmental Effects
6.1.
Overview of
Solvent
Polarity Effects
....................... 205
6.1.1.
Effects of Solvent Polarity
............................ 205
6.1.2.
Polarity Surrounding a Membrane-Bound
Fluorophore
................................................... 206
6.1.3.
Other Mechanisms for Spectral Shifts
.......... 207
6.2.
General Solvent Effects: The Lippert-Mataga
Equation
................................................................... 208
6.2.1.
Derivation of the Lippert Equation
............... 210
6.2.2.
Application of the Lippert Equation
............. 212
6.3.
Specific Solvent Effects
........................................... 213
6.3.1.
Specific Solvent Effects and Lippert Plots
... 215
6.4.
Temperature Effects
................................................. 216
6.5.
Phase Transitions in Membranes
............................. 217
6.6.
Additional Factors that Affect Emission Spectra
..... 219
6.6.1.
Locally Excited and Internal
Charge-Transfer States
.................................. 219
6.6.2.
Excited-State Intramolecular Proton
Transfer (ESIPT)
........................................... 221
6.6.3.
Changes in the Non-Radiative
Decay Rates
................................................... 222
6.6.4.
Changes in the Rate of Radiative Decay
...... 223
6.7.
Effects of Viscosity
.................................................. 223
6.7.1.
Effect of Shear Stress on Membrane
Viscosity
........................................................ 225
6.8.
Probe-Probe Interactions
......................................... 225
6.9.
Biochemical Applications of Environment-
Sensitive Fluorophores
............................................. 226
6.9.1.
Fatty-Acid-Binding Proteins
......................... 226
6.9.2.
Exposure of
a
Hydrophobie
Surface
on Calmodulin
............................................... 226
6.9.3.
Binding to Cyclodextrin Using a
Dansyl Probe
................................................. 227
6.10.
Advanced Solvent-Sensitive Probes
......................... 228
6.11.
Effects of Solvent Mixtures
...................................... 229
6.12.
Summary of Solvent Effects
..................................... 231
References
................................................................ 232
Problems
................................................................... 235
7.
Dynamics of Solvent and Spectral Relaxation
7.1.
Overview of Excited-State Processes
....................... 237
7.1.1.
Time-Resolved Emission Spectra
................. 239
7.2.
Measurement of Time-Resolved Emission
Spectra
(TRES)
........................................................ 240
7.2.1.
Direct Recording of
TRES
............................ 240
7.2.2.
TRES
from Wavelength-Dependent
Decays
........................................................... 241
7.3.
Spectral Relaxation in Proteins
................................ 242
7.3.1.
Spectral Relaxation of Labeled
Apomyoglobin
............................................... 243
7.3.2.
Protein Spectral Relaxation around a
Synthetic Fluorescent
Amino
Acid
............... 244
7.4.
Spectral Relaxation in Membranes
.......................... 245
7.4.1.
Analysis of Time-Resolved Emission
Spectra
........................................................... 246
7.4.2.
Spectral Relaxation of Membrane-Bound
Anthroyloxy Fatty Acids
............................... 248
7.5.
Picosecond Relaxation in Solvents
.......................... 249
7.5.1.
Theory for Time-Dependent Solvent
Relaxation
...................................................... 250
7.5.2.
Multi-Exponential Relaxation in Water
........ 251
7.6.
Measurement of Multi-Exponential Spectral
Relaxation
................................................................. 252
7.7.
Distinction between Solvent Relaxation
and Formation of Rotational Isomers
...................... 253
7.8.
Comparison of
TRES
and Decay-Associated
Spectra
...................................................................... 255
7.9.
Lifetime-Resolved Emission Spectra
....................... 255
7.10.
Red-Edge Excitation Shifts
...................................... 257
7.10.1.
Membranes and Red-Edge
Excitation Shifts
.......................................... 258
7.10.2.
Red-Edge Excitation Shifts and
Energy Transfer
........................................... 259
7.11.
Excited-State Reactions
............................................ 259
7.11.1.
Excited-State Ionization of Naphthol
.......... 260
7.12.
Theory for a Reversible Two-State Reaction
........... 262
7.12.1.
Steady-State Fluorescence of a
Two-State Reaction
..................................... 262
7.12.2.
Time-Resolved Decays for the
Two-State Model
......................................... 263
7.12.3.
Differential Wavelength Methods
............... 264
7.13.
Time-Domain Studies of Naphthol Dissociation
..... 264
7.14.
Analysis of Excited-State Reactions by
Phase-Modulation Fluorometry
................................ 265
7.14.1.
Effect of an Excited-State Reaction
on the Apparent Phase and Modulation
Lifetimes
...................................................... 266
7.14.2.
Wavelength-Dependent Phase and
Modulation Values for an Excited-State
Reaction
....................................................... 267
7.14.3.
Frequency-Domain Measurement of
Excimer Formation
...................................... 269
7.15.
Biochemical Examples of Excited-State
Reactions
.................................................................. 270
7.15.1.
Exposure of a Membrane-Bound
Cholesterol Analogue
.................................. 270
References
................................................................ 270
Problems
................................................................... 275
8.
Quenching of Fluorescence
8.1.
Quenchers of Fluorescence
...................................... 278
8.2.
Theory of CoUisional Quenching
............................. 278
8.2.1.
Derivation of the
Stern-Volmer
Equation
..... 280
8.2.2.
Interpretation of the Bimolecular
Quenching Constant
...................................... 281
8.3.
Theory of Static Quenching
..................................... 282
8.4.
Combined Dynamic and Static Quenching
.............. 282
8.5.
Examples of Static and Dynamic Quenching
.......... 283
8.6.
Deviations from the Stern-
Volmer
Equation:
Quenching Sphere of Action
.................................... 284
8.6.1.
Derivation of the Quenching Sphere
of Action
........................................................ 285
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
8.7.
Effects of Steric Shielding and Charge on
Quenching
................................................................ 286
8.7.1.
Accessibility of DNA-Bound Probes
to Quenchers
.................................................. 286
8.7.2.
Quenching of Ethenoadenine Derivatives
..... 287
8.8.
Fractional Accessibility to Quenchers
...................... 288
8.8.1.
Modified Stern-
Volmer
Plots
........................ 288
8.8.2.
Experimental Considerations
in Quenching
................................................. 289
8.9.
Applications of Quenching to Proteins
.................... 290
8.9.1.
Fractional Accessibility of Tryptophan
Residues in Endonuclease III
........................ 290
8.9.2.
Effect of Conformational Changes
on Tryptophan Accessibility
.......................... 291
8.9.3.
Quenching of the Multiple Decay
Times of Proteins
.......................................... 291
8.9.4.
Effects of Quenchers on Proteins
.................. 292
8.9.5.
Correlation of Emission Wavelength
and Accessibility: Protein Folding of
ColicinEl
...................................................... 292
8.10.
Application of Quenching to Membranes
................ 293
8.10.1.
Oxygen Diffusion in Membranes
................ 293
8.10.2.
Localization of Membrane-Bound
Tryptophan Residues by Quenching
........... 294
8.10.3.
Quenching of Membrane Probes
Using Localized Quenchers
........................ 295
8.10.4.
Parallax and Depth-Dependent
Quenching in Membranes
........................... 296
8.10.5.
Boundary
Lipid
Quenching
......................... 298
8.10.6.
Effect of Lipid-Water Partitioning
on Quenching
.............................................. 298
8.10.7.
Quenching in Micelles
................................ 300
8.11.
Lateral Diffusion in Membranes
.............................. 300
8.12.
Quenching-Resolved Emission Spectra
................... 301
8.12.1.
Fluorophore Mixtures
.................................. 301
8.12.2.
Quenching-Resolved Emission Spectra
of the
E. Coli
Tet Repressor
........................ 302
8.13.
Quenching and Association Reactions
..................... 304
8.13.1.
Quenching Due to Specific Binding
Interactions
.................................................. 304
8.14.
Sensing Applications of Quenching
......................... 305
8.14.1.
Chloride-Sensitive Fluorophores
................. 306
8.14.2.
Intracellular Chloride Imaging
.................... 306
8.14.3.
Chloride-Sensitive GFP
............................... 307
8.14.4.
Amplified Quenching
.................................. 309
8.15.
Applications of Quenching to Molecular
Biology
..................................................................... 310
8.15.1.
Release of Quenching upon
Hybridization
............................................... 310
8.15.2.
Molecular Beacons in Quenching
by Guanine
.................................................. 311
8.15.3.
Binding of Substrates to Ribozymes
........... 311
8.15.4.
Association Reactions and Accessibility
to Quenchers
................................................ 312
8.16.
Quenching on Gold Surfaces
.................................... 313
8.16.1.
Molecular Beacons Based on Quenching
by Gold Colloids
......................................... 313
8.16.2.
Molecular Beacons Based on Quenching
by a Gold Surface
........................................ 314
8.17.
Intramolecular Quenching
........................................ 314
8.17.1. DNA
Dynamics by Intramolecular
Quenching
................................................... 314
8.17.2.
Electron-Transfer Quenching in a
Flavoprotein
................................................. 315
8.17.3.
Sensors Based on Intramolecular
PET Quenching
........................................... 316
8.18.
Quenching of Phosphorescence
............................... 317
References
................................................................ 318
Problems
................................................................... 327
9.
Mechanisms and Dynamics of
Fluorescence Quenching
9.1.
Comparison of Quenching and Resonance
Energy Transfer
........................................................ 331
9.1.1.
Distance Dependence of RET
and Quenching
.............................................. 332
9.1.2.
Encounter Complexes and Quenching
Efficiency
...................................................... 333
9.2.
Mechanisms of Quenching
....................................... 334
9.2.1.
Intersystem Crossing
..................................... 334
9.2.2.
Electron-Exchange Quenching
...................... 335
9.2.3.
Photoinduced Electron Transfer
.................... 335
9.3.
Energetics of Photoinduced Electron Transfer
........ 336
9.3.1.
Examples of PET Quenching
........................ 338
9.3.2.
PET in Linked Donor-Acceptor Pairs
.......... 340
9.4.
PET Quenching in Biomolecules
............................. 341
9.4.1.
Quenching of
Indole
by Imidazolium
........... 341
9.4.2.
Quenching by
DNA
Bases and
Nucleotides
.................................................... 341
9.5.
Single-Molecule PET
............................................... 342
9.6.
Transient Effects in Quenching
................................ 343
9.6.1.
Experimental Studies of Transient
Effects
............................................................ 346
9.6.2.
Distance-Dependent Quenching
in Proteins
...................................................... 348
References
................................................................ 348
Problems
................................................................... 351
1
0.
Fluorescence Anisotropy
10.1.
Definition of Fluorescence Anisotropy
.................... 353
10.1.1.
Origin of the Definitions of
Polarization and Anisotropy
........................ 355
10.2.
Theory for Anisotropy
.............................................. 355
10.2.1.
Excitation Photoselection of Fluorophores
. 357
10.3.
Excitation Anisotropy Spectra
.................................. 358
10.3.1.
Resolution of Electronic States from
Polarization Spectra
.................................... 360
10.4.
Measurement of Fluorescence Anisotropies
............ 361
10.4.1.
L-Format or Single-Channel Method
.......... 361
10.4.2.
Т
-Format
or Two-Channel Anisotropies
...... 363
10.4.3.
Comparison of
Т
-Format
and
L-Format Measurements
............................. 363
CONTENTS
10.4.4.
Alignment of Polarizers
............................... 364
10.4.5.
Magic-Angle Polarizer Conditions
............. 364
10.4.6.
Why is the Total Intensity
Equal to
/„ +
2IL
.......................................... 364
10.4.7.
Effect of Resonance Energy Transfer
on the Anisotropy
........................................ 364
10.4.8.
Trivial Causes of Depolarization
................. 365
10.4.9.
Factors Affecting the Anisotropy
................ 366
10.5.
Effects of Rotational Diffusion on Fluorescence
Anisotropies: The Perrin Equation
........................... 366
10.5.1.
The Perrin Equation: Rotational
Motions of Proteins
..................................... 367
10.5.2.
Examples of a Perrin Plot
........................... 369
10.6.
Perrin Plots of Proteins
............................................. 370
10.6.1.
Binding of tRNA to tRNA Synthetase
........ 370
10.6.2.
Molecular Chaperonin
српбО
(GroEL)
....... 371
10.6.3.
Perrin Plots of an Fab Immunoglobulin
Fragment
...................................................... 371
10.7.
Biochemical Applications of Steady-State
Anisotropies
.............................................................. 372
10.7.1.
Peptide
Binding to Calmodulin
................... 372
10.7.2.
Binding of the
Trp
Repressor
to
DNA........ 373
10.7.3. Helicase-Catalyzed DNA
Unwinding
......... 373
10.7.4.
Melittin Association Detected from
Homotransfer
............................................... 374
10.8.
Anisotropy of Membranes and Membrane-
Bound Proteins
......................................................... 374
10.8.1.
Membrane Microviscosity
........................... 374
10.8.2.
Distribution of Membrane-Bound
Proteins
........................................................ 375
10.9.
Transition Moments
.................................................. 377
References
................................................................ 378
Additional Reading on the Application
of Anisotropy
...................................................... 380
Problems
................................................................... 381
I I
.
Time-Dependent Anisotropy Decays
11.1.
Time-Domain and Frequency-Domain
Anisotropy Decays
................................................... 383
11.2.
Anisotropy Decay Analysis
...................................... 387
11.2.1.
Early Methods for Analysis of
TD Anisotropy Data
.................................... 387
11.2.2.
Preferred Analysis of TD
Anisotropy Data
.......................................... 388
11.2.3.
Value of r0
.................................................... 389
11.3.
Analysis of Frequency-Domain
Anisotropy Decays
................................................... 390
11.4.
Anisotropy Decay Laws
........................................... 390
11.4.1.
Non-Spherical Fluorophores
....................... 391
11.4.2.
Hindered Rotors
.......................................... 391
11.4.3.
Segmental
Mobility of a Biopolymer-
Bound Fluorophore
..................................... 392
11.4.4.
Correlation Time Distributions
................... 393
11.4.5.
Associated Anisotropy Decays
.................... 393
11.4.6.
Example Anisotropy Decays of
Rhodamine Green and Rhodamine
Green-Dextran
............................................. 394
11.5.
Time-Domain Anisotropy Decays of Proteins
......... 394
11.5.1.
Intrinsic Tryptophan Anisotropy Decay
of Liver Alcohol Dehydrogenase
................ 395
11.5.2.
Phospholipase A2
......................................... 395
11.5.3.
Subtilisin
Carlsberg
..................................... 395
11.5.4.
Domain Motions of Immunoglobulins
........ 396
11.5.5.
Effects of Free Probe on Anisotropy
Decays
......................................................... 397
11.6.
Frequency-Domain Anisotropy Decays
of Proteins
................................................................. 397
11.6.1.
Apomyoglobin: A Rigid Rotor
.................... 397
11.6.2.
Melittin Self-Association and
Anisotropy Decays
...................................... 398
11.6.3.
Picosecond Rotational Diffusion
of Oxytocin
.................................................. 399
11.7.
Hindered Rotational Diffusion in Membranes
......... 399
11.7.1.
Characterization of a New
Membrane Probe
......................................... 401
11.8.
Anisotropy Decays of Nucleic Acids
....................... 402
11.8.1.
Hydrodynamics of
DNA
Oligomers...........
403
11.8.2.
Dynamics of Intracellular
DNA.................. 403
11.8.3. DNA
Binding to
HIV Integrase
Using
Correlation Time Distributions
................... 404
11.9.
Correlation Time Imaging
........................................ 406
11.10.
Microsecond Anisotropy Decays
............................ 408
11.10.1.
Phosphorescence Anisotropy Decays
........ 408
11.10.2.
Long-Lifetime Metal-Ligand
Complexes
................................................. 408
References
................................................................ 409
Problems
................................................................... 412
12.
Advanced Anisotropy Concepts
12.1.
Associated Anisotropy Decay
................................... 413
12.1.1.
Theory for Associated Anisotropy
Decay
........................................................... 414
12.1.2.
Time-Domain Measurements of
Associated Anisotropy Decays
.................... 415
12.2.
Biochemical Examples of Associated
Anisotropy Decays
................................................... 417
12.2.1.
Time-Domain Studies of
DNA
Binding to the Klenow Fragment
of
DNA Polymerase.................................... 417
12.2.2.
Frequency-Domain Measurements
of Associated Anisotropy Decays
............... 417
12.3.
Rotational Diffusion of Non-Spherical
Molecules: An Overview
.......................................... 418
12.3.1.
Anisotropy Decays of Ellipsoids
................. 419
12.4.
Ellipsoids of Revolution
........................................... 420
12.4.1.
Simplified Ellipsoids of Revolution
............ 421
12.4.2.
Intuitive Description of Rotational
Diffusion of an Oblate Ellipsoid
................. 422
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
12.4.3.
Rotational Correlation Times for
Ellipsoids of Revolution
.............................. 423
12.4.4.
Stick-versus-Slip Rotational Diffusion
....... 425
12.5.
Complete Theory for Rotational Diffusion
of Ellipsoids
.............................................................. 425
12.6. Anisotropie
Rotational Diffusion
............................. 426
12.6.1.
Time-Domain Studies
.................................. 426
12.6.2.
Frequency-Domain Studies of
Anisotropie
Rotational Diffusion
................ 427
12.7.
Global Anisotropy Decay Analysis
.......................... 429
12.7.1.
Global Analysis with Multi-Wavelength
Excitation
.................................................... 429
12.7.2.
Global Anisotropy Decay Analysis with
Collisional Quenching
................................. 430
12.7.3.
Application of Quenching to Protein
Anisotropy Decays
...................................... 431
12.8.
Intercalated Fluorophores in
DNA........................... 432
12.9.
Transition Moments
.................................................. 433
12.9.1.
Anisotropy of Planar Fluorophores
with High Symmetry
................................... 435
12.10.
Lifetime-Resolved Anisotropies
............................. 435
12.10.1.
Effect of
Segmental
Motion on the
Perrin Plots
.............................................. 436
12.11.
Soleillet s Rule: Multiplication of Depolarized
Factors
.................................................................... 436
12.12.
Anisotropies Can Depend on Emission
Wavelength
............................................................. 437
References
.............................................................. 438
Problems
................................................................. 441
13.
Energy Transfer
13.1.
Characteristics of Resonance Energy Transfer
........ 443
13.2.
Theory of Energy Transfer for a
Donor-Acceptor Pair
................................................ 445
13.2.1.
Orientation Factor
к2
................................... 448
13.2.2.
Dependence of the Transfer Rate on
Distance
0),
the Overlap
Integral
(/),
and
τ2
....................................... 449
13.2.3.
Homotransfer
and
Heterotransfer................
450
13.3.
Distance Measurements Using
RET........................ 451
13.3.1.
Distance Measurements in
α
-Helical
Melittin
........................................................ 451
13.3.2.
Effects of Incomplete Labeling
................... 452
13.3.3.
Effect of
к2
on the Possible Range
of Distances
................................................. 452
13.4.
Biochemical Applications of RET
........................... 453
13.4.1.
Protein Folding Measured by RET
............. 453
13.4.2.
Intracellular Protein Folding
....................... 454
13.4.3.
RET and Association Reactions
.................. 455
13.4.4.
Orientation of a Protein-Bound
Peptide
...... 456
13.4.5.
Protein Binding to Semiconductor
Nanoparticles
............................................... 457
13.5.
RET Sensors
............................................................. 458
13.5.1.
Intracellular RET Indicator
for Estrogens
............................................... 458
13.5.2.
RET Imaging of Intracellular Protein
Phosphorylation
........................................... 459
13.5.3.
Imaging of
Rac
Activation in Cells
............. 459
13.6.
RET and Nucleic Acids
............................................ 459
13.6.1.
Imaging of Intracellular
RNA
..................... 460
13.7.
Energy-Transfer Efficiency from
Enhanced Acceptor Fluorescence
............................. 461
13.8.
Energy Transfer in Membranes
................................ 462
13.8.1.
Lipid
Distributions around Gramicidin
....... 463
13.8.2.
Membrane Fusion and
Lipid
Exchange
...... 465
13.9.
Effect of
к2
on RET
.................................................. 465
13.10.
Energy Transfer in Solution
................................... 466
13.10.1.
Diffusion-Enhanced Energy Transfer
........ 467
13.11.
Representative Ro Values
........................................ 467
References
................................................................ 468
Additional References on Resonance
Energy Transfer
................................................... 471
Problems
................................................................... 472
1
4.
Time-Resolved Energy Transfer and
Conformational Distributions of
Biopolymers
14.1.
Distance Distributions
.............................................. 477
14.2.
Distance Distributions in Peptides
........................... 479
14.2.1.
Comparison for a Rigid and Flexible
Hexapeptide
................................................. 479
14.2.2.
Crossfitting
Data to Exclude
Alternative Models
...................................... 481
14.2.3.
Donor Decay without Acceptor
.................. 482
14.2.4.
Effect of Concentration of the
D
-А
Pairs
.................................................... 482
14.3.
Distance Distributions in Peptides
........................... 482
14.3.1.
Distance Distributions in Melittin
............... 483
14.4.
Distance-Distribution Data Analysis
........................ 485
14.4.1.
Frequency-Domain Distance-Distribution
Analysis
....................................................... 485
14.4.2.
Time-Domain Distance-Distribution
Analysis
....................................................... 487
14.4.3.
Distance-Distribution Functions
................. 487
14.4.4.
Effects of Incomplete Labeling
................... 487
14.4.5.
Effect of the Orientation Factor
к2
.............. 489
14.4.6.
Acceptor Decays
.......................................... 489
14.5.
Biochemical Applications of Distance
Distributions
............................................................. 490
14.5.1.
Calcium-Induced Changes in the
Conformation of Troponin
С
...................... 490
14.5.2.
Hairpin Ribozyme
....................................... 493
14.5.3.
Four-Way Holliday Junction in
DNA......... 493
14.5.4.
Distance Distributions and Unfolding
of Yeast Phosphoglycerate Kinase
.............. 494
14.5.5.
Distance Distributions in a Glycopeptide
... 495
14.5.6.
Single-Protein-Molecule Distance
Distribution
.................................................. 496
14.6.
Time-Resolved RET Imaging
................................... 497
14.7.
Effect of Diffusion for Linked
D
-А
Pairs
................ 498
CONTENTS
14.7.1.
Simulations
of FRET for a Flexible
D-APair
...................................................... 499
14.7.2.
Experimental Measurement of D-A
Diffusion for a Linked
D
-А
Pair
................ 500
14.7.3.
FRET and Diffusive Motions in
Biopolymers................................................ 501
14.8.
Conclusion
................................................................ 501
References
................................................................ 501
Representative Publications on Measurement
of Distance Distributions
.................................... 504
Problems
................................................................... 505
15.
Energy Transfer to Multiple Acceptors in
One.Two, or Three Dimensions
15.1.
RET in Three Dimensions
........................................ 507
15.1.1.
Effect of Diffusion on FRET with
Unlinked Donors and Acceptors
................. 508
15.1.2.
Experimental Studies of RET in
Three Dimensions
....................................... 509
15.2.
Effect of Dimensionality on RET
............................ 511
15.2.1.
Experimental FRET in Two Dimensions
.... 512
15.2.2.
Experimental FRET in One Dimension
...... 514
15.3.
Biochemical Applications of RET with
Multiple Acceptors
................................................... 515
15.3.1.
Aggregation of
β
-Amyloid Peptides
........... 515
15.3.2.
RET Imaging of Fibronectin
....................... 516
15.4.
Energy Transfer in Restricted Geometries
............... 516
15.4.1.
Effect of Excluded Area on Energy
Transfer in Two Dimensions
....................... 518
15.5.
RET in the Presence of Diffusion
............................ 519
15.6.
RET in the Rapid Diffusion Limit
........................... 520
15.6.1.
Location of an Acceptor in
Lipid
Vesicles
.............................................. 521
15.6.2.
Locaion of Retinal in Rhodopsin
Disc Membranes
.......................................... 522
15.7.
Conclusions
.............................................................. 524
References
................................................................ 524
Additional References on RET between
Unlinked Donor and Acceptor
............................ 526
Problems
................................................................... 527
16.
Protein Fluorescence
16.1.
Spectral Properties of the Aromatic
Amino
Acids...
530
16.1.1.
Excitation Polarization Spectra of
Tyrosine and Tryptophan
............................. 531
16.1.2.
Solvent Effects on Tryptophan Emission
Spectra
......................................................... 533
16.1.3.
Excited-State Ionization of Tyrosine
........... 534
16.1.4.
Tyrosinate Emission from Proteins
............. 535
16.2.
General Features of Protein Fluorescence
................ 535
16.3.
Tryptophan Emission in an
Apoiar
Protein Environment
................................................. 538
16.3.1.
Site-Directed Mutagenesis of a
Single-Tryptophan Azurin
........................... 538
16.3.2.
Emission Spectra of Azurins with
One or Two Tryptophan Residues
............... 539
16.4.
Energy Transfer and Intrinsic Protein
Fluorescence
............................................................. 539
16.4.1.
Tyrosine-to-Tryptophan Energy Transfer
in
Interferon
-γ
..............................................
540
16.4.2.
Quantitation of RET Efficiencies
in Proteins
.................................................... 541
16.4.3.
Tyrosine-to-Tryptophan RET in
a Membrane-Bound Protein
........................ 543
16.4.4.
Phenylalanine-to-Tyrosine
Energy Transfer
........................................... 543
16.5.
Calcium Binding to Calmodulin Using
Phenylalanine and Tyrosine Emission
...................... 545
16.6.
Quenching of Tryptophan Residues in Proteins
....... 546
16.6.1.
Effect of Emission Maximum on
Quenching
................................................... 547
16.6.2.
Fractional Accessibility to Quenching
in Multi-Tryptophan Proteins
...................... 549
16.6.3.
Resolution of Emission Spectra by
Quenching
................................................... 550
16.7.
Association Reaction of Proteins
............................. 551
16.7.1.
Binding of Calmodulin to a
Target Protein
.............................................. 551
16.7.2.
Calmodulin: Resolution of the
Four Calcium-Binding Sites Using
Tryptophan-Containing Mutants
................. 552
16.7.3.
Interactions of
DNA
with Proteins
.............. 552
16.8.
Spectral Properties of Genetically Engineered
Proteins
..................................................................... 554
16.8.1.
S
ingle-Try ptophan Mutants of
Triosephosphate Isomerase
......................... 555
16.8.2.
Barnase: A Three-Tryptophan Protein
........ 556
16.8.3.
Site-Directed Mutagenesis of
Tyrosine Proteins
......................................... 557
16.9.
Protein Folding
......................................................... 557
16.9.1.
Protein Engineering of Mutant
Ribonuclease
f
or Folding Experiments
....... 558
16.9.2.
Folding of
Lactate
Dehydrogenase
............. 559
16.9.3.
Folding Pathway of CRABPI
...................... 560
16.10.
Protein Structure and Tryptophan Emission
.......... 560
16.10.1.
Tryptophan Spectral Properties
and Structural Motifs
............................... 561
16.11.
Tryptophan Analogues
............................................ 562
16.11.1.
Tryptophan Analogues
............................. 564
16.11.2.
Genetically Inserted Amino-Acid
Analogues
................................................ 565
16.12.
The Challenge of Protein Fluorescence
................. 566
References
.............................................................. 567
Problems
................................................................. 573
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
1
7.
Time-Resolved Protein Fluorescence
17.1.
Intensity Decays of Tryptophan:
The Rotamer Model
................................................. 578
17.2.
Time-Resolved Intensity Decays of
Tryptophan and Tyrosine
.......................................... 580
17.2.1.
Decay-Associated Emission Spectra
of Tryptophan
.............................................. 581
17.2.2.
Intensity Decays of Neutral Tryptophan
Derivatives
................................................... 581
17.2.3.
Intensity Decays of Tyrosine and
Its Neutral Derivatives
................................. 582
17.3.
Intensity and Anisotropy Decays of Proteins
........... 583
17.3.1.
Single-Exponential Intensity and
Anisotropy Decay of Ribonuclease T,
........ 584
17.3.2. Annexin
V: A Calcium-Sensitive
Single-Tryptophan Protein
.......................... 585
17.3.3.
Anisotropy Decay of a Protein with
Two Tryptophans
......................................... 587
17.4.
Protein Unfolding Exposes the Tryptophan
Residue to Water
....................................................... 588
17.4.1.
Conformational Heterogeneity Can
Result in Complex Intensity and
Anisotropy Decays
...................................... 588
17.5.
Anisotropy Decays of Proteins
................................. 589
17.5.1.
Effects of Association Reactions on
Anisotropy Decays: Melittin
....................... 590
17.6.
Biochemical Examples Using Time-Resolved
Protein Fluorescence
................................................ 591
17.6.1.
Decay-Associated Spectra of Barnase
........ 591
17.6.2.
Disulfide Oxidoreductase DsbA
................. 591
17.6.3.
Immunophilin FKBP59-I: Quenching
of Tryptophan Fluorescence by
Phenylalanine
.............................................. 592
17.6.4.
Trp
Repressor:
Resolution of the Two
Interacting Tryptophans
.............................. 593
17.6.5.
Thermophilic
ß-Glycosidase:
A Multi-Tryptophan Protein
........................ 594
17.6.6.
Heme
Proteins Display Useful
Intrinsic Fluorescence
................................. 594
17.7.
Time-Dependent Spectral Relaxation of
Tryptophan
................................................................ 596
17.8.
Phosphorescence of Proteins
.................................... 598
17.9.
Perspectives on Protein Fluorescence
...................... 600
References
................................................................ 600
Problems
................................................................... 605
1
8.
Multiphoton Excitation and Microscopy
18.1.
Introduction to Multiphoton Excitation
................... 607
18.2.
Cross-Sections for Multiphoton Absorption
............ 609
18.3.
Two-Photon Absorption Spectra
............................... 609
18.4.
Two-Photon Excitation of a DNA-Bound
Fluorophore
.............................................................. 610
18.5.
Anisotropies with Multiphoton Excitation
............... 612
18.5.1.
Excitation Photoselection for
Two-Photon Excitation
................................ 612
18.5.2.
Two-Photon Anisotropy of
DPH
................. 612
18.6.
МРЕ
for a Membrane-Bound Fluorophore
.............. 613
18.7.
МРЕ
of Intrinsic Protein Fluorescence
.................... 613
18.8.
Multiphoton Microscopy
.......................................... 616
18.8.1.
Calcium Imaging
......................................... 616
18.8.2.
Imaging of NAD(P)H and FAD
.................. 617
18.8.3.
Excitation of Multiple Fluorophores
........... 618
18.8.4.
Three-Dimensional Imaging of Cells
.......... 618
References
................................................................ 619
Problems
................................................................... 621
19.
Fluorescence Sensing
19.1.
Optical Clinical Chemistry and Spectral
Observables
.............................................................. 623
19.2.
Spectral
Observables
for Fluorescence Sensing
....... 624
19.2.1.
Optical Properties of Tissues
...................... 625
19.2.2.
Lifetime-Based Sensing
.............................. 626
19.3.
Mechanisms of Sensing
............................................ 626
19.4.
Sensing by Collisional Quenching
........................... 627
19.4.1.
Oxygen Sensing
.......................................... 627
19.4.2.
Lifetime-Based Sensing of Oxygen
............ 628
19.4.3.
Mechanism of Oxygen Selectivity
.............. 629
19.4.4.
Other Oxygen Sensors
................................ 629
19.4.5.
Lifetime Imaging of Oxygen
...................... 630
19.4.6.
Chloride Sensors
......................................... 631
19.4.7.
Lifetime Imaging of Chloride
Concentrations
............................................. 632
19.4.8.
Other Collisional Quenchers
....................... 632
19.5.
Energy-Transfer Sensing
.......................................... 633
19.5.1. pH
and pCO2 Sensing by
Energy Transfer
........................................... 633
19.5.2.
Glucose Sensing by Energy Transfer
.......... 634
19.5.3.
Ion Sensing by Energy Transfer
.................. 635
19.5.4.
Theory for Energy-Transfer Sensing
........... 636
19.6.
Two-State
pH
Sensors
.............................................. 637
19.6.1.
Optical Detection of Blood Gases
.............. 637
19.6.2. pH
Sensors
.................................................. 637
19.7.
Photoinduced Electron Transfer (PET) Probes
for Metal Ions and
Anion
Sensors
............................ 641
19.8.
Probes of Analyte Recognition
................................. 643
19.8.1.
Specificity of Cation Probes
....................... 644
19.8.2.
Theory of Analyte Recognition Sensing
..... 644
19.8.3.
Sodium and Potassium Probes
.................... 645
19.8.4.
Calcium and Magnesium Probes
................ 647
19.8.5.
Probes for Intracellular Zinc
....................... 650
19.9.
Glucose-Sensitive Fluorophores
............................... 650
19.10.
Protein Sensors
....................................................... 651
19.10.1.
Protein Sensors Based on RET
................ 652
19.11.
GFP Sensors
........................................................... 654
19.11.1.
GFP Sensors Using RET
.......................... 654
19.11.2.
Intrinsic GFP Sensors
............................... 655
xxiv
CONTENTS
19.12. New
Approaches to Sensing
................................... 655
19.12.1.
Pebble Sensors and Lipobeads
................. 655
19.13.
In-Vivo Imaging
..................................................... 656
19.14.
Immunoassays
........................................................ 658
19.14.1.
Enzyme-Linked Immunosorbent Assays
(ELISA)
................................................... 659
19.14.2.
Time-Resolved Immunoassays
................ 659
19.14.3.
Energy-Transfer Immunoassays
.............. 660
19.14.4.
Fluorescence Polarization
Immunoassays
......................................... 661
References
.............................................................. 663
Problems
................................................................. 672
20.
Novel Fluorophores
20.1.
Semiconductor Nanoparticles
................................... 675
20.1.1.
Spectral Properties of QDots
...................... 676
20.1.2.
Labeling Cells with QDots
.......................... 677
20.1.3.
QDots and Resonance Energy Transfer
...... 678
20.2. Lanthanides............................................................... 679
20.2.1.
RET with
Lanthanides................................ 680
20.2.2. Lanthanide
Sensors
..................................... 681
20.2.3. Lanthanide
Nanoparticles
............................ 682
20.2.4.
Near-Infrared Emitting
Lanthanides........... 682
20.2.5. Lanthanides
and Fingerprint Detection
....... 683
20.3.
Long-Lifetime Metal-Ligand Complexes
................ 683
20.3.1.
Introduction to Metal-Ligand Probes
......... 683
20.3.2.
Anisotropy Properties of
Metal-Ligand Complexes
........................... 685
20.3.3.
Spectral Properties of MLC Probes
............ 686
20.3.4.
The Energy Gap Law
.................................. 687
20.3.5.
Biophysical Applications of
Metal-Ligand Probes
.................................. 688
20.3.6.
MLC Immunoassays
................................... 691
20.3.7.
Metal-Ligand Complex Sensors
................. 694
20.4.
Long-Wavelength Long-Lifetime
Fluorophores
............................................................. 695
References
................................................................ 697
Problems
................................................................... 702
21. DNA
Technology
21.1. DNA
Sequencing
...................................................... 705
21.1.1.
Principle of
DNA
Sequencing
..................... 705
21.1.2.
Examples of
DNA
Sequencing
................... 706
21.1.3.
Nucleotide Labeling Methods
..................... 707
21.1.4.
Example of
DNA
Sequencing
..................... 708
21.1.5.
Energy-Transfer Dyes for
DNA
Sequencing
.................................................. 709
21.1.6. DNA
Sequencing with NIR Probes
............ 710
21.1.7. DNA
Sequencing Based on Lifetimes
........ 712
21.2.
High-Sensitivity
DNA
Stains
................................... 712
21.2.1.
High-Affinity
Bis DNA
Stains
.................... 713
21.2.2.
Energy-Transfer
DNA
Stains
...................... 715
21.2.3. DNA
Fragment Sizing by
Flow Cytometry
........................................... 715
21.3. DNA
Hybridization
.................................................. 715
21.3.1. DNA
Hybridization Measured with
One-Donor- and Acceptor-Labeled
DNA
Probe
.................................................. 717
21.3.2. DNA
Hybridization Measured by
Excimer Formation
...................................... 718
21.3.3.
Polarization Hybridization Arrays
.............. 719
21.3.4.
Polymerase Chain Reaction
........................ 720
21.4.
Molecular Beacons
................................................... 720
21.4.1.
Molecular Beacons with
Nonfluorescent Acceptors
........................... 720
21.4.2.
Molecular Beacons with
Fluorescent Acceptors
................................. 722
21.4.3.
Hybridization Proximity Beacons
............... 722
21.4.4.
Molecular Beacons Based on
Quenching by Gold
..................................... 723
21.4.5.
Intracellular Detection of mRNA
Using Molecular Beacons
........................... 724
21.5.
Aptamers
................................................................... 724
21.5.1.
DNAzymes
.................................................. 726
21.6.
Multiplexed Microbead Arrays:
Suspension Arrays
.................................................... 726
21.7.
Fluorescence In-Situ Hybridization
......................... 727
21.7.1.
Preparation of FISH Probe
DNA................ 728
21.7.2.
Applications of FISH
.................................. 729
21.8.
Multicolor FISH and Spectral Karyotyping
............. 730
21.9. DNA
Arrays
.............................................................. 732
21.9.1.
Spotted
DNA
Microarrays
.......................... 732
21.9.2.
Light-Generated
DNA
Arrays
..................... 734
References
................................................................ 734
Problems
................................................................... 740
22.
Fluorescence-Lifetime Imaging Microscopy
22.1.
Early Methods for Fluorescence-Lifetime
Imaging
..................................................................... 743
22.1.1.
FLIM Using Known Fluorophores
............. 744
22.2.
Lifetime Imaging of Calcium Using Quin-2
............ 744
22.2.1.
Determination of Calcium Concentration
from Lifetime
.............................................. 744
22.2.2.
Lifetime Images of Cos Cells
..................... 745
22.3.
Examples of Wide-Field Frequency-Domain
FLIM
......................................................................... 746
22.3.1.
Resonance Energy-Transfer FLIM
of Protein Kinase
С
Activation
................... 746
22.3.2.
Lifetime Imaging of Cells Containing
TwoGFPs
.................................................... 7^7-
22.4.
Wide-Field FLIM Using a Gated-Image
Intensifier
.................................................................. 747
22.5.
Laser Scanning TCSPC FLIM
................................. 748
22.5.1.
Lifetime Imaging of Cellular
Biomolecules
............................................... 750
22.5.2.
Lifetime Images of Amyloid Plaques
......... 750
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
22.6.
Frequency-Domain Laser Scanning Microscopy
.....
22.7.
Conclusions
..............................................................
References
................................................................
Additional Reading on Fluorescence-Lifetime
Imaging Microscopy
...........................................
Problem
.....................................................................
23.
Single-Molecule Detection
23.1.
Detectability of Single Molecules
...........................
23.2.
Total Internal Reflection and Confocal Optics
........
23.2.1.
Total Internal Reflection
.............................
23.2.2.
Confocal Detection Optics
.........................
23.3.
Optical Configurations for SMD
.............................
23.4.
Instrumentation for SMD
........................................
23.4.1.
Detectors for Single-Molecule Detection
..
23.4.2.
Optical Filters for SMD
.............................
23.5.
Single-Molecule Photophysics
................................
23.6.
Biochemical Applications of SMD
.........................
23.6.1.
Single-Molecule Enzyme Kinetics
.............
23.6.2.
Single-Molecule ATPase Activity
..............
23.6.3.
Single-Molecule Studies of a
Chaperonin Protein
.....................................
23.7.
Single-Molecule Resonance Energy Transfer
.........
23.8.
Single-Molecule Orientation and Rotational
Motions
....................................................................
23.8.1.
Orientation Imaging of R6G and GFP
.......
23.8.2.
Imaging of
Dipole
Radiation Patterns
........
23.9.
Time-Resolved Studies of Single Molecules
..........
23.10.
Biochemical Applications
......................................
23.10.1.
Turnover of Single Enzyme Molecules..
23.10.2.
Single-Molecule Molecular Beacons
.....
23.10.3.
Conformational Dynamics of a
Holliday Junction
...................................
23.10.4.
Single-Molecule Calcium Sensor
...........
23.10.5.
Motions of Molecular Motors
................
23.11.
Advanced Topics in SMD
......................................
23.1
1
.1.
Signal-to-Noise Ratio in
Single-Molecule Detection
.....................
23.11.2.
Polarization of Single Immobilized
Fluorophores
...........................................
23.11.3.
Polarization Measurements
and Mobility of Surface-Bound
Fluorophores
...........................................
23.11.4.
Single-Molecule Lifetime Estimation
...,
23.12.
Additional Literature on SMD
..............................
References
.............................................................
Additional References on Single-Molecule
Detection
..........................................................
Problem
..................................................................
24.
Fluorescence Correlation Spectroscopy
24.1.
Principles of Fluorescence Correlation
Spectroscopy
............................................................
750 24.2.
Theory of FCS
.......................................................... 800
752 24.2.1.
Translational Diffusion and FCS
................. 802
752 24.2.2.
Occupation Numbers and Volumes
in FCS
.......................................................... 804
753 24.2.3.
FCS for Multiple Diffusing Species
........... 804
755 24.3.
Examples of FCS Experiments
................................ 805
24.3.1.
Effect of Fluorophore Concentration
.......... 805
24.3.2.
Effect of Molecular Weight on
Diffusion Coefficients
................................. 806
24.4.
Applications of FCS to Bioaffinity Reactions
.......... 807
24.4.1.
Protein Binding to the
Chaperonin GroEL
...................................... 807
24.4.2.
Association of Tubulin Subunits
................. 807
24.4.3. DNA
Applications of FCS
.......................... 808
24.5.
FCS in Two Dimensions: Membranes
..................... 810
24.5.1.
Biophysical Studies of Lateral
Diffusion in Membranes
............................. 812
24.5.2.
Binding to Membrane-Bound
Receptors
..................................................... 813
24.6.
Effects of Intersystem Crossing
............................... 815
24.6.1.
Theory for FCS and Intersystem
Crossing
....................................................... 816
24.7.
Effects of Chemical Reactions
................................. 816
24.8.
Fluorescence Intensity Distribution Analysis
........... 817
24.9.
Time-Resolved FCS
................................................. 819
24.10.
Detection of Conformational Dynamics
in Macromolecules
................................................. 820
24.11.
FCS with Total Internal Reflection
........................ 821
24.12.
FCS with Two-Photon Excitation
........................... 822
24.12.1.
Diffusion of an Intracellular
Kinase Using FCS with
Two-Photon Excitation
............................ 823
24.13.
Dual-Color Fluorescence Cross-Correlation
Spectroscopy
........................................................... 823
24.13.
1
.
Instrumentation for Dual-Color
FCCS
....................................................... 824
24.13.2.
Theory of Dual-Color FCCS
................... 824
24.13.3. DNA
Cleavage by a
Restriction Enzyme
................................. 826
24.13.4.
Applications of Dual-Color FCCS
.......... 826
24.14.
Rotational Diffusion and Photo Antibunching
....... 828
24.15.
Flow Measurements Using FCS
............................. 830
24.16.
Additional References on FCS
............................... 832
References
.............................................................. 832
Additional References to FCS and
Its Applications
................................................. 837
Problems
................................................................. 840
759
760
760
761
762
764
765
766
768
770
770
770
771
773
775
777
778
779
780
780
782
782
784
784
784
784
786
786
787
788
788
791
795
25.
Radiative Decay Engineering:
Metal-Enhanced Fluorescence
25.1.
Radiative Decay Engineering
................................... 841
25.1.1.
Introduction to RDE
.................................... 841
25.1.2.
Jabłoński
Diagram for Metal-
Enhanced Fluorescence
............................... 842
798 25.2.
Review of Metal Effects on Fluorescence
................ 843
25.3.
Optical Properties of Metal Colloids
....................... 845
25.4.
Theory for Fluorophore-Colloid Interactions
.......... 846
25.5.
Experimental Results on Metal-Enhanced
Fluorescence
............................................................. 848
25.5.1.
Application of MEF to
DNA
Analysis
........ 848
25.6.
Distance-Dependence of Metal-Enhanced
Fluorescence
............................................................. 851
25.7.
Applications of Metal-Enhanced Fluorescence
........ 851
25.7.1. DNA
Hybridization Using MEF
................. 853
25.7.2.
Release of Self-Quenching
.......................... 853
25.7.3.
Effect of Silver Particles on RET
................ 854
25.8.
Mechanism of MEF
.................................................. 855
25.9.
Perspective on RET
.................................................. 856
References
................................................................ 856
Problem
..................................................................... 859
26.
Radiative Decay Engineering:
Surface Plasmon-Coupled Emission
26.1.
Phenomenon of SPCE
.............................................. 861
26.2.
Surface-Plasmon Resonance
.................................... 861
26.2.1.
Theory for Surface-Plasmon Resonance
..... 863
26.3.
Expected Properties of SPCE
................................... 865
26.4.
Experimental Demonstration of SPCE
..................... 865
26.5.
Applications of SPCE
............................................... 867
26.6.
Future Developments in SPCE
................................. 868
References
................................................................ 870
Appendix I. Corrected Emission Spectra
1.
Emission Spectra Standards from
300
to
800
nm
......... 873
2. ß-Carboline
Derivatives as Fluorescence Standards
..... 873
3.
Corrected Emission Spectra of 9,10-Diphenyl-
anthracene, Quinine, and Fluorescein
........................... 877
4.
Long-Wavelength Standards
.......................................... 877
5.
Ultraviolet Standards
..................................................... 878
6.
Additional Corrected Emission Spectra
........................ 881
References
..................................................................... 881
CONTENTS
Appendix II. Fluorescent Lifetime Standards
1.
Nanosecond Lifetime Standards
.................................... 883
2.
Picosecond Lifetime Standards
..................................... 884
3.
Representative Frequency-Domain
Intensity Decays
............................................................ 885
4.
Time-Domain Lifetime Standards
................................. 886
Appendix III. Additional Reading
1.
Time-Resolved Measurements
.................................... 889
2.
Spectra Properties of Fluorophores
............................. 889
3.
Theory of Fluorescence and Photophysics
.................. 889
4.
Reviews of Fluorescence Spectroscopy
...................... 889
5.
Biochemical Fluorescence
.......................................... 890
6.
Protein Fluorescence
................................................... 890
7.
Data Analysis and Nonlinear Least Squares
............... 890
8.
Photochemistry
............................................................ 890
9.
Flow Cytometry
........................................................... 890
10.
Phosphorescence
.......................................................... 890
11.
Fluorescence Sensing
.................................................. 890
12.
Immunoassays
............................................................. 891
13.
Applications of Fluorescence
...................................... 891
14.
Multiphoton Excitation
................................................ 891
15.
Infrared and NIR Fluorescence
................................... 891
16.
Lasers
........................................................................... 891
17.
Fluorescence Microscopy
............................................ 891
18.
Metal—Ligand Complexes and Unusual
Lumophores
................................................................. 891
19.
Single-Molecule Detection
.......................................... 891
20.
Fluorescence Correlation Spectroscopy
...................... 892
21.
Biophotonics
................................................................ 892
22.
Nanoparticles
............................................................... 892
23.
Metallic Particles
......................................................... 892
24.
Books on Fluorescence
................................................ 892
Answers to Problems
..................... 893
Index
.................................... 923
|
| any_adam_object | 1 |
| author | Lakowicz, Joseph R. |
| author_facet | Lakowicz, Joseph R. |
| author_role | aut |
| author_sort | Lakowicz, Joseph R. |
| author_variant | j r l jr jrl |
| building | Verbundindex |
| bvnumber | BV036867319 |
| classification_rvk | UH 5870 VG 8750 |
| ctrlnum | (OCoLC)700510097 (DE-599)BSZ333381904 |
| dewey-full | 543.56 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 543 - Analytical chemistry |
| dewey-raw | 543.56 |
| dewey-search | 543.56 |
| dewey-sort | 3543.56 |
| dewey-tens | 540 - Chemistry and allied sciences |
| discipline | Chemie / Pharmazie Physik |
| edition | 3. ed. (corr. at 4. print.) |
| format | Book |
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| id | DE-604.BV036867319 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T22:49:46Z |
| institution | BVB |
| isbn | 0387312781 9780387312781 9781489978806 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-020782951 |
| oclc_num | 700510097 |
| open_access_boolean | |
| owner | DE-703 DE-355 DE-BY-UBR DE-20 DE-83 |
| owner_facet | DE-703 DE-355 DE-BY-UBR DE-20 DE-83 |
| physical | XXVI, 954 S. Ill., zahlr. graph. Darst. |
| publishDate | 2010 |
| publishDateSearch | 2010 |
| publishDateSort | 2010 |
| publisher | Springer |
| record_format | marc |
| spelling | Lakowicz, Joseph R. Verfasser aut Principles of fluorescence spectroscopy Joseph R. Lakowicz 3. ed. (corr. at 4. print.) New York, NY Springer 2010 XXVI, 954 S. Ill., zahlr. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Hier auch später erschienene, unveränderte Nachdrucke Fluoreszenz (DE-588)4154818-8 gnd rswk-swf Kinetik (DE-588)4030665-3 gnd rswk-swf Fluoreszenzspektroskopie (DE-588)4017701-4 gnd rswk-swf Spektrometer (DE-588)4140820-2 gnd rswk-swf Organische Verbindungen (DE-588)4043816-8 gnd rswk-swf Fluoreszenzspektroskopie (DE-588)4017701-4 s SWB Spektrometer (DE-588)4140820-2 s 1\p DE-604 Fluoreszenz (DE-588)4154818-8 s 2\p DE-604 Organische Verbindungen (DE-588)4043816-8 s 3\p DE-604 Kinetik (DE-588)4030665-3 s 4\p DE-604 Erscheint auch als Online-Ausgabe 978-0-387-46312-4 Digitalisierung UB Bayreuth application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020782951&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 4\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Lakowicz, Joseph R. Principles of fluorescence spectroscopy Fluoreszenz (DE-588)4154818-8 gnd Kinetik (DE-588)4030665-3 gnd Fluoreszenzspektroskopie (DE-588)4017701-4 gnd Spektrometer (DE-588)4140820-2 gnd Organische Verbindungen (DE-588)4043816-8 gnd |
| subject_GND | (DE-588)4154818-8 (DE-588)4030665-3 (DE-588)4017701-4 (DE-588)4140820-2 (DE-588)4043816-8 |
| title | Principles of fluorescence spectroscopy |
| title_auth | Principles of fluorescence spectroscopy |
| title_exact_search | Principles of fluorescence spectroscopy |
| title_full | Principles of fluorescence spectroscopy Joseph R. Lakowicz |
| title_fullStr | Principles of fluorescence spectroscopy Joseph R. Lakowicz |
| title_full_unstemmed | Principles of fluorescence spectroscopy Joseph R. Lakowicz |
| title_short | Principles of fluorescence spectroscopy |
| title_sort | principles of fluorescence spectroscopy |
| topic | Fluoreszenz (DE-588)4154818-8 gnd Kinetik (DE-588)4030665-3 gnd Fluoreszenzspektroskopie (DE-588)4017701-4 gnd Spektrometer (DE-588)4140820-2 gnd Organische Verbindungen (DE-588)4043816-8 gnd |
| topic_facet | Fluoreszenz Kinetik Fluoreszenzspektroskopie Spektrometer Organische Verbindungen |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020782951&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT lakowiczjosephr principlesoffluorescencespectroscopy |