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Contents :
1. Emerging Biomedical Applications of Time-Resolved Fluorescence
Spectroscopy
Joseph R. Lakowicz
1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2. Schemes for Fluorescence Sensing . . . . . . . . . . . . . . . . . 2
1.2.1. Instrument Complexity, Measurement Scheme,
and the Spectral Properties of Fluorophores . . . . . . . . 4
1.2.2. Lifetime-Based Sensing. . . . . . . . . . . . . . . . . . . 5
1.3. Applications of Fluorescence to Clinical Sensing . . . . . . . . . 7
1.3.1. Phase-Modulation Sensing of Blood Gases
and/or Blood Septicemia . . . . . . . . . . . . . . . . . . 7
1.3.2. Noninvasive Transdermal Glucose Sensing . . . . . . . . . . 8
1.4. Applications to Cell Biology and Physiology. . . . . . . . . . . .12
1.4.1. Intracellular Chemical Analysis and Flow Cytometry. . . . .12
1.4.2. Fluorescence Lifetime Imaging Microscopy (FLIM) . . . . . .13
1.5. Conclusion: The Need for Development of New Fluorescence Probes. .17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
2. Principles of Fluorescent Probe Design for Ion Recognition
Bernard Valeur
2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .21
2.2. Fluorescent Signaling Receptors of Cations . . . . . . . . . . . .23
2.2.1. Fundamental Aspects . . . . . . . . . . . . . . . . . . . .23
2.2.2. Recognition Based on Cation Control of
Photoinduced Electron Transfer in
Nonconjugated Donor-Acceptor Systems. . . . . . . . . . . .25
2.2.3. Recognition Based on Cation Control of
Photoinduced Charge Transfer in Conjugated
Donor-Acceptor Systems. . . . . . . . . . . . . . . . . . .28
2.2.4. Recognition Based on Cation Control of the
Proximity between Two Fluorophores, or a
Fluorophore and a Quencher. . . . . . . . . . . . . . . . .37
2.2.5. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . .41
2.3. Fluorescent Signaling Receptors of Anions. . . . . . . . . . . . .42
2.4. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . .44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
3. Fluorescent Chemosensors for Cations, Anions, and Neutral Analytes
Anthony W. Czarnik
3.1. Chelation-Enhanced Fluorescence in 9,10-Bis(TMEDA)anthracene . . .51
3.2. Chelation-Enhanced Fluorescence of Anthlylazamacrocycle
Chemosensors in Aqueous Solution . . . . . . . . . . . . . . . . .53
3.3. Chelatoselective Fluorescence Perturbation in an
Anthlylazamacrocycle CHEF Sensor . . . . . . . . . . . . . . . . .57
3.4. Chelation-Enhanced Fluorescence Detection of Nonmetal Ions . . . .59
3.5. An Assay for Enzyme-CataIyzed Polyanion Hydrolysis Based
on Template-Directed Excimer Formation . . . . . . . . . . . . . .62
3.6. Fluorescence Chemosensing of Carbohydrates . . . . . . . . . . . .66
3.7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
4. Design and Applications of Highly Luminescent Transition Metal
Complexes
J. N. Demas and B. A. DeGraff
4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .71
4.2. States of Inorganic Complexes. . . . . . . . . . . . . . . . . . .74
4.3. Design Considerations. . . . . . . . . . . . . . . . . . . . . . .76
4.4. Temperature Effects on Inorganic Sensors . . . . . . . . . . . . .78
4.5. Design Examples. . . . . . . . . . . . . . . . . . . . . . . . . .81
4.6. Sensor Design and Applications . . . . . . . . . . . . . . . . . .85
4.6.1. Probe/Sensor Design . . . . . . . . . . . . . . . . . . . .85
4.6.2. Applications. . . . . . . . . . . . . . . . . . . . . . . .89
4.7. Microheterogenous Systems. . . . . . . . . . . . . . . . . . . . .92
4.7.1. Simulations . . . . . . . . . . . . . . . . . . . . . . . .93
4.7.2. Uniqueness: A Caveat. . . . . . . . . . . . . . . . . . . .95
4.7.3. Simulation Results. . . . . . . . . . . . . . . . . . . . .97
4.7.4. Physical System Results . . . . . . . . . . . . . . . . . 100
4.8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . 103
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5. Fluorescence Probes Based on Twisted Intramolecular Charge
Transfer(TICT)States and Other Adiabatic Photoreactions
W. Rettig and Rene Lapouyade
5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . l09
5.2. Adiabatic Photochemical Reaction Mechanisms or How to
Produce Large Stokes Shifts. . . . . . . . . . . . . . . . . . . 111
5.2.1. Twisting and Charge Transfer: The TICT Mechanism. . . . . 113
5.2.2. Intramolecular Proton Transfer: The ESIPT Mechanism . . . 114
5.2.3. Intramolecular Folding: The Excimer/Exciplex
Mechanism and Dewar Isomerization
(Butterfly Mechanism) . . . . . . . . . . . . . . . . . . 117
5.3. Examples of Polarity Probes. . . . . . . . . . . . . . . . . . . 118
5.4. Examples of Free Volume Probes . . . . . . . . . . . . . . . . . 120
5.4.1. Excimer Probes. . . . . . . . . . . . . . . . . . . . . . 122
5.4.2. UCT Probes. . . . . . . . . . . . . . . . . . . . . . . . 122
5.4.3. Butterfly Probes. . . . . . . . . . . . . . . . . . . . . 124
5.5. How to Construct Proton- and Ion-Sensitive Analytical Probes:
Principles and General Scheme of Use . . . . . . . . . . . . . . 125
5.5. 1. Generating Sensitivity through Introduction
of TICT-Pathways . . . . . . . . . . . . . . . . . . . . 125
5.5.2. General Use as Indicators . . . . . . . . . . . . . . . . 127
5.6. pH Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.6.1. Physiological pH Indicators . . . . . . . . . . . . . . . 129
5.6.2. Fluorescent Probes with an Efficient Intramolecular
Fluorescence Quenching Process in the Base Form
Possibly Related to the Formation of a Nonemissive
TICT State. . . . . . . . . . . . . . . . . . . . . . . . 129
5.6.3. Donor-Acceptor and Donor-Donor Substitution Stilbenes . . 131
5.6.4. "Fluor-Spacer-Receptor" Systems with a
Photoinduced Electron Transfer as a Quenching
Process of the Fluorescence . . . . . . . . . . . . . . . 133
5.7. Ion Complexing Probes. . . . . . . . . . . . . . . . . . . . . . 135
5.7.1. Monoaza-15-Crown-5 Stilbenes Forming
Emissive TICT States. . . . . . . . . . . . . . . . . . . 135
5.7.2. Fluorescent Calcium Indicators in Current Use in
Molecular Biology . . . . . . . . . . . . . . . . . . . . 136
5.7.3. Other Fluoroionophores with Enhanced
Fluorescence in the Presence of Cations . . . . . . . . . 139
5.8. Basic Ideas for Future Developments. . . . . . . . . . . . . . . 140
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6. Red and Near-Infrared Fluorometry
Richard B. Thompson
6.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . 151
6.2. Background and Rationale. . . . . . . . . . . . . . . . . . . . 151
6.3. Excitation Sources. . . . . . . . . . . . . . . . . . . . . . . 152
6.3.1. Gas and Dye Lasers . . . . . . . . . . . . . . . . . . . 153
6.3.2. Light-Emitting Diodes. . . . . . . . . . . . . . . . . . 154
6.3.3. Titanium:Sapphire Lasers . . . . . . . . . . . . . . . . 155
6.3.4. Diode Lasers . . . . . . . . . . . . . . . . . . . . . . 158
6.3.5. External Modulation. . . . . . . . . . . . . . . . . . . 162
6.4. Detectors and Optics. . . . . . . . . . . . . . . . . . . . . . 163
6.4.1. Photomultiplier Tubes. . . . . . . . . . . . . . . . . . 163
6.4.2. Photodiodes and Avalanche Photodiodes. . . . . . . . . . 165
6.4.3. Infrared Optics. . . . . . . . . . . . . . . . . . . . . 166
6.5. Infrared Fluorophores . . . . . . . . . . . . . . . . . . . . . 167
6.5.1. Cyanines . . . . . . . . . . . . . . . . . . . . . . . . 168
6.5.2. Oxazines . . . . . . . . . . . . . . . . . . . . . . . . 171
6.5.3. Polynuclear Aromatic Hydrocarbons. . . . . . . . . . . . 172
6.5.4. Phthalocyanines. . . . . . . . . . . . . . . . . . . . . 173
6.5.5. Other Infrared Fluorophores. . . . . . . . . . . . . . . 174
6.6. Scattering, Absorbance, and Interfering Fluorescence. . . . . . 175
6.7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 178
References. . . . . . . . . . . . . . . . . . . . . . . . . . . 179
7. Near-Infrared Fluorescence Probes
Guillermo A. Casay, Dana B. Shealy, and Gabor Patonay
7.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . 183
7.1.1. Background . . . . . . . . . . . . . . . . . . . . . . . 184
7.1.2. Characteristics of the NIR Region. . . . . . . . . . . . 186
7.2. NIR Optical Probe Instrumentation . . . . . . . . . . . . . . . 187
7.2.1. Light Sources. . . . . . . . . . . . . . . . . . . . . . 189
7.2.2. Detection. . . . . . . . . . . . . . . . . . . . . . . . 191
7.2.3. Miscellaneous Components . . . . . . . . . . . . . . . . 194
7.2.4. Optical Probe. . . . . . . . . . . . . . . . . . . . . . 195
7.3. Optical Fiber Measurements. . . . . . . . . . . . . . . . . . . 206
7.3.1. Metal Ion Determination. . . . . . . . . . . . . . . . . 206
7.3.2. Solution pH Determination. . . . . . . . . . . . . . . . 209
7.3.3. Biosensors . . . . . . . . . . . . . . . . . . . . . . . 211
7.4. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . 216
References. . . . . . . . . . . . . . . . . . . . . . . . . . . 217
8. Fluorescence Spectroscopy in Turbid Media and Tissues
Dieter Oelkrug
8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 223
8.2. Basic Photometric Quantities . . . . . . . . . . . . . . . . . . 224
8.3. Experimental Methods . . . . . . . . . . . . . . . . . . . . . . 225
8.3.1. Conventional Fluorimeters . . . . . . . . . . . . . . . . 225
8.3.2. Diode Array Spectrometers . . . . . . . . . . . . . . . . 227
8.3.3. Time-Resolved Measurements. . . . . . . . . . . . . . . . 228
8.3.4. Locally Resolved Measurements . . . . . . . . . . . . . . 231
8.3.5. Diffuse Reflectance Spectra of Fluorescent Samples. . . . 232
8.4. Model Calculations . . . . . . . . . . . . . . . . . . . . . . . 233
8.4.1. Solution of the Equations of Transfer . . . . . . . . . . 235
8.4.2. Spot Irradiation. . . . . . . . . . . . . . . . . . . . . 236
8.4.3. Extended Area of Irradiation. . . . . . . . . . . . . . . 237
8.4.4. Time-Resolved Analysis. . . . . . . . . . . . . . . . . . 241
8.5. Determination of Scattering and Absorption Coefficients. . . . . 243
8.6. Quantitative Fluorescence Analysis . . . . . . . . . . . . . . . 246
8.6.1. Forward and Backward Fluorescence . . . . . . . . . . . . 246
8.6.2. Inner Filter Effects. . . . . . . . . . . . . . . . . . . 248
8.6.3. Fluorescence Reabsorption . . . . . . . . . . . . . . . . 248
8.6.4. Fluorescence Quantum Yields . . . . . . . . . . . . . . . 250
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
9. Real-Ilme Chemical Sensing Employing Luminescence Techniques
J. Ricardo Alcala
9.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 255
9.2. Basic Principles . . . . . . . . . . . . . . . . . . . . . . . . 256
9.2.1. Homogeneous Sensors . . . . . . . . . . . . . . . . . . . 256
9.2.2. Luminescence and Sensing. . . . . . . . . . . . . . . . . 259
9.2.3. Nonhomogeneous Sensors. . . . . . . . . . . . . . . . . . 260
9.3. Continuous Wave Luminescence Sensing . . . . . . . . . . . . . . 263
9.3.1. Homogeneous Sensors . . . . . . . . . . . . . . . . . . . 263
9.3.2. Nonhomogeneous Sensors. . . . . . . . . . . . . . . . . . 264
9.4. Time-Resolved Luminescence Sensing . . . . . . . . . . . . . . . 264
9.4.1. Homogeneous Sensors . . . . . . . . . . . . . . . . . . . 265
9.4.2. Nonhomogeneous Sensors. . . . . . . . . . . . . . . . . . 265
9.5. Real-Time Techniques . . . . . . . . . . . . . . . . . . . . . . 269
9.5.1. The Nature of Intensity and Lifetime-Based Sensors. . . . 270
9.5.2. Time Domain and Frequency Domain Measurements . . . . . . 270
9.5.3. The Principle of Frequency Domain Sensing . . . . . . . . 272
9.5.4. Concurrent Multifrequency Measurements. . . . . . . . . . 276
9.5.5. The Limit of Fourier Methods in Real-Time Sensing . . . . 283
9.5.6. Noise in the Time and in the Frequency Domain . . . . . . 283
9.5.7. Fiberoptic Sensor Instrumentation . . . . . . . . . . . . 284
9.6. Example: An Oxygen Sensor. . . . . . . . . . . . . . . . . . . . 288
9.7. Example: A Temperature Sensor. . . . . . . . . . . . . . . . . . 291
9.8. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
10. Lifetime-Based Sensing
Henryk Szmacinski and Joseph R. Lakowicz
10.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 295
10.2. Requirements of a Fluorescent Indicator. . . . . . . . . . . . 299
10.3. Molecular Mechanisms for Fluorescence Lifetime-Based Sensing . 301
10.4. Measurement of Fluorescence Lifetimes. . . . . . . . . . . . . 304
10.5. Sensing Based on Probe-Analyte Recognition . . . . . . . . . . 307
10.5.1. Intensity-Based Sensing. . . . . . . . . . . . . . . . 308
10.5.2. Lifetime-Based Sensing . . . . . . . . . . . . . . . . 311
10.6. Sensing Based on Collisional Quenching of Fluorescence . . . . 317
10.6.1. Oxygen Sensing . . . . . . . . . . . . . . . . . . . . 317
10.6.2. Cellular Chloride Sensing. . . . . . . . . . . . . . . 319
10.7. Sensing Based on Fluorescence Resonance
Energy Transfer (FRET) . . . . . . . . . . . . . . . . . . . . 321
10.7.l. Unlinked Donor-Acceptor. . . . . . . . . . . . . . . . 322
10.7.2. Linked Donor-Acceptor. . . . . . . . . . . . . . . . . 324
10.7.3. Macromolecules Labeled by Donor and Acceptor . . . . . 327
10.8. Summary and Prospects. . . . . . . . . . . . . . . . . . . . . 328
References . . . . . . . . . . . . . . . . . . . . . . . . . . 329
11. Fiber Optic Fluorescence Thermometry
K. T. V. Grattan and Z. Y. Zhang
11.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 335
11.1.1. Fiber Optic Temperature Measurement. . . . . . . . . . 335
11.1.2. Fiber Optic Sensor Devices for Temperature
Measurement. . . . . . . . . . . . . . . . . . . . . . 337
11.2. Fluorescence-Based Fiber Optic Thermometry . . . . . . . . . . 338
11.2.1. Photoluminescence in Fiber Optic Thermometry . . . . . 338
11.2.2. Classes of Fluorescent Materials for
Fluorescence Thermometry . . . . . . . . . . . . . . . 338
11.2.3. Early Fluorescence Thermometer Schemes . . . . . . . . 339
11.2.4. Fluorescence Lifetime-Based Schemes. . . . . . . . . . 342
11.2.5. Pulse Measurement of Fluorescence Lifetime . . . . . . 342
11.2.6. Phase and Modulation Measurement . . . . . . . . . . . 347
11.2.7. Phase-Locked Detection of Fluorescence Lifetime. . . . 348
11.3. Solid-State Materials for Fluorescence Thermometry . . . . . . 351
11.3.1. Cr3+-Based Material. . . . . . . . . . . . . . . . . . 351
11.3.2. Optical Arrangement of Fluorescence
Lifetime Thermometers. . . . . . . . . . . . . . . . . 355
11.3.3. Ruby-Based Thermometer with Range from 20
to 600 C deg.. . . . . . . . . . . . . . . . . . . . . 358
11.3.4. Alexandrite-Based Thermometer with Range
-100-700 C deg.. . . . . . . . . . . . . . . . . . . . 360
11.3.5. Cr:LiSAF-Based Thermometer for Biomedical
Applications . . . . . . . . . . . . . . . . . . . . . 363
11.3.6. Discussion of Cr3+ Doping Effects in Thermometry . . . 365
11.3.7. Cross-Referencing of Fluorescence Thermometry with
Blackbody Radiation Pyrometry. . . . . . . . . . . . . 366
11.4. Discussion and Cross-Comparison of Experimental Devices. . . . 370
11.4.1. Cross-Comparison . . . . . . . . . . . . . . . . . . . 370
11.4.2. Assessment of Fiber Optic Thermometers . . . . . . . . 371
References . . . . . . . . . . . . . . . . . . . . . . . . . . 373
12. Instrumentation for Red/Near-Infrared Fluorescence
David J. S. Birch and Graham Hungerford
12.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 377
12.2. Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 378
12.2.1. Steady-State Spectra . . . . . . . . . . . . . . . . . 378
12.2.2. Lifetimes. . . . . . . . . . . . . . . . . . . . . . . 380
12.2.3. Anisotropy . . . . . . . . . . . . . . . . . . . . . . 383
12.2.4. Microscopy . . . . . . . . . . . . . . . . . . . . . . 384
12.2.5. Multiwavelength Array Detection. . . . . . . . . . . . 386
12.2.6. Sensors. . . . . . . . . . . . . . . . . . . . . . . . 386
12.2.7. High-Performance Liquid Chromatography . . . . . . . . 390
12.3. Sources. . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
12.3.1. Lamps. . . . . . . . . . . . . . . . . . . . . . . . . 391
12.3.2. Flashlamps . . . . . . . . . . . . . . . . . . . . . . 392
12.3.3. Light-Emitting Diodes. . . . . . . . . . . . . . . . . 395
12.3.4. Diode Lasers . . . . . . . . . . . . . . . . . . . . . 397
12.3.5. Other Sources. . . . . . . . . . . . . . . . . . . . . 399
12.4. Detectors. . . . . . . . . . . . . . . . . . . . . . . . . . . 401
12.4.1. Photomultipliers . . . . . . . . . . . . . . . . . . . 402
12.4.2. Microchannel Plate Photomultipliers. . . . . . . . . . 404
12.4.3. Streak Cameras . . . . . . . . . . . . . . . . . . . . 406
12.4.4. Photodiodes. . . . . . . . . . . . . . . . . . . . . . 406
12.4.5. Avalanche Photodiodes. . . . . . . . . . . . . . . . . 409
12.5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . 411
References . . . . . . . . . . . . . . . . . . . . . . . . . . 411
13. Application of Fluorescence Sensing to Bioreactors
Govind Rao, Shabbir B. Bambot, Simon C. W. Kwong,
Henryk Szmacinski, Jeffrey Sipior, Raja Holavanahali, and Gary Carter
13.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 417
13.2. Dissolved Oxygen Sensing . . . . . . . . . . . . . . . . . . . 419
13.3. pH Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . 421
13.4. pCO2 Sensing . . . . . . . . . . . . . . . . . . . . . . . . . 422
13.5. Glucose Sensing. . . . . . . . . . . . . . . . . . . . . . . . 422
13.6. Off-Gas Analysis . . . . . . . . . . . . . . . . . . . . . . . 423
13.7. Biomass Concentration. . . . . . . . . . . . . . . . . . . . . 424
13.8. Culture Fluorescence . . . . . . . . . . . . . . . . . . . . . 424
13.8.l. Biomass Estimation . . . . . . . . . . . . . . . . . . 425
13.8.2. Substrate Addition/Depletion Responses . . . . . . . . 425
13.8.3. Aerobic-Anaerobic Transitions. . . . . . . . . . . . . 425
13.9. Other Approaches . . . . . . . . . . . . . . . . . . . . . . . 428
13.10.The Future . . . . . . . . . . . . . . . . . . . . . . . . . . 428
13.10.1. Cost Considerations . . . . . . . . . . . . . . . . . 431
13.10.2. Fluorescence Lifetime-Based Oxygen Sensor . . . . . . 432
13.10.3. pH Sensors. . . . . . . . . . . . . . . . . . . . . . 437
13.10.4. Glucose Sensors . . . . . . . . . . . . . . . . . . . 438
13.10.5. Utilization of Low-Cost Red LED and
Laser Diode Sources . . . . . . . . . . . . . . . . . 440
References . . . . . . . . . . . . . . . . . . . . . . . . . . 444
14. Principles of Fluorescence Immunoassay
Alvydas J. Ozinskas
14.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 449
14.2. Fluorescence Inimunoassay Reagents . . . . . . . . . . . . . . 450
14.2.1. Antibodies . . . . . . . . . . . . . . . . . . . . . . 451
14.2.2. Fluorescent Probes . . . . . . . . . . . . . . . . . . 452
14.3. Fluorescence Instrumentation . . . . . . . . . . . . . . . . . 456
14.4. Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . 457
14.5. Fluorescence Immunoassay Applications. . . . . . . . . . . . . 460
14.5.1. Fluorescence Polarization Immunoassays . . . . . . . . 461
14.5.2. Time-Resolved Fluorescence Immunoassays. . . . . . . . 465
14.5.3. Fluorescence Energy Transfer Immunoassays. . . . . . . 469
14.5.4. Phase-Modulation Fluoroimmunoassays. . . . . . . . . . 473
14.5.5. Liposome Fluoroimmunoassays. . . . . . . . . . . . . . 482
14.5.6. Fluoroimmunosensors. . . . . . . . . . . . . . . . . . 484
14.6. Discussion and Conclusions . . . . . . . . . . . . . . . . . . 488
References . . . . . . . . . . . . . . . . . . . . . . . . . . 490
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
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