Fluorescence Applications in Biotechnology and Life Sciences

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ISBN: 9780470083703 | 0470083700
Cover: Hardcover
Copyright: 8/24/2009

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This is the first reference in this important subject area to focus on fundamental concepts and applications of fluorescence in biotechnology and life sciences. The book emphasizes the principles and focus on the 'here and now' rather than the 'over the horizon' research which might become available in the future. The book also raises awareness of scientific approaches, technologies, and applications that may help to resolve industrially and medically relevant problems in areas such as public health, food processing and safety, and environmental monitoring.

Preface	p. xv
Acknowledgments	p. xxi
About the Contributing Authors	p. xxiii
Basics of Fluorescence	p. 1
Introduction	p. 1
Properties of Light	p. 2
States of Molecules	p. 5
Absorption and Emission of Light	p. 9
Interaction of Light with a Molecule	p. 9
Absorption and Emission of Light Depicted on a Perrin-Jablonski Diagram	p. 10
Multiphoton Excitation	p. 11
Noradiative Decay Mechanisms	p. 13
Vibrational Relaxation	p. 13
Internal Conversion	p. 14
Intersystem Crossing	p. 14
Properties of Excited Molecules	p. 16
Quantum Yield	p. 16
Excited-State Lifetime	p. 16
Spectroscopy and Fluorophores	p. 16
Absorption, Excitation, and Emission Spectra	p. 16
Basic Properties of Fluorophores	p. 19
Environmental Sensitivity of Fluorophores	p. 20
Quenching of Fluorescence	p. 21
Photobleaching	p. 21
Fluorescence Resonance Energy Transfer	p. 22
Solvent Relaxation	p. 22
Polarization of Fluorescence	p. 24
Conclusion	p. 26
References	p. 26
Labeling of Cells with Fluorescent Dyes	p. 27
Introduction	p. 27
Fluorophores Selection	p. 32
Loading and Labeling Live Cells	p. 34
Loading AM Forms	p. 35
Leakage and Hydrolysis of AM Esters	p. 36
Fluorophores for Live Cell Imaging	p. 36
Cell Viability	p. 36
General Morphology	p. 36
Specific Organelles	p. 37
Ionic Environment	p. 38
Tracers for Membranes and Cells	p. 39
Fluorescent Proteins	p. 40
References	p. 41
Genetically Encoded Fluorescent Probes: Some Properties and Applications in Life Sciences	p. 47
Introduction	p. 47
Chromophore and its Formation	p. 49
"Life and Death" of Fluorescent Protein	p. 54
Oligomerization	p. 57
Fusion Proteins	p. 58
Photobleaching	p. 58
Destabilization	p. 59
Applications	p. 60
Passive Applications	p. 61
Photoamplifying PAFPs	p. 62
Photoconverting PAFPs	p. 63
Kindling PAFPs	p. 64
Fluorescent Timers	p. 64
Active Applications	p. 65
Protein-Protein Interactions	p. 65
Monitoring Enzyme Activity	p. 66
Monitoring Small Molecules and Metabolites	p. 66
Monitoring pH and Other Small Molecules	p. 67
Monitoring Redox	p. 67
Interactive Applications	p. 67
Conclusions	p. 68
References	p. 69
Nanoparticle Fluorescence Probes	p. 75
Introduction	p. 75
Nanomaterials for Biological Applications	p. 75
Inorganic Quantum Dots: Physics and Optical Properties	p. 77
Synthesis of Monodisperse Colloidal Quantum Dots for Biolabeling Applications	p. 81
Quantum Dot Synthesis	p. 82
Shell Synthesis	p. 84
Water Solubilization and Functionalization	p. 85
Quantum Dot Bioconjugation	p. 88
Quantum Dots as In Vitro Probes	p. 89
Quantum Dots as In Vivo Probes	p. 92
Cytotoxicity	p. 94
Future Directions	p. 94
References	p. 95
Quantitative Analysis of Fluorescent Image: From Descriptive to Computational Microscopy	p. 99
Introduction	p. 99
Advantages of Quantitative Analysis	p. 100
Methods of Quantitative Analysis	p. 101
Image Processing	p. 113
References	p. 115
Spectral Imaging and Unmixing	p. 117
Introduction	p. 117
Instrumentation and Configurations for Hyperspectral Microscopes	p. 119
Supervised and Unsupervised Unmixing	p. 121
Supervised or Informed Unmixing	p. 121
Unsupervised or Blind Unmixing	p. 122
Iterated Constrained End-Members Algorithm	p. 123
Spectral Clustering	p. 124
Examples	p. 125
Tablet Inspection	p. 125
Fluorescent Microspheres	p. 127
Induced Mouse Lung Tumor	p. 131
Limitations of Spectral Imaging and Experimental Considerations	p. 135
Fast Biological Processes	p. 135
Calibration	p. 136
Environmental Sensitivity	p. 137
Background Fluorescence	p. 137
Conclusions	p. 137
References	p. 138
Correlation of Light with Electron Microscopy: A Correlative Microscopy Platform	p. 141
Introduction	p. 141
Overview of Techniques Used in Correlative Microscopy	p. 142
Correlative Microscopy of Chemically Fixed, Immunolabeled Ultrathin Cryosections Employing Antibodies Coupled with Fluorescent Gold Conjugates	p. 145
Special Techniques Used in Sample Preparation for Correlative Microscopy: High-Pressure Freezing, Freeze Fracturing, and Freeze Substitution	p. 151
Correlative Microscopy of Fixed Tissue Specimens Using Freeze Substitution	p. 153
Future Trends: Correlative Microscopy and High-Content Cellular Screening	p. 154
Future Trends: Correlative Microscopy of Fluorescent Images Acquired by Confocal Laser Scanning Microscopy	p. 154
References	p. 155
Fluorescence Resonance Energy Transfer and Applications	p. 157
What is FRET?	p. 157
Theory of FRET	p. 157
Why FRET Can Be Useful	p. 159
How FRET Can Be Measured	p. 160
Applications of FRET in Spectroscopy	p. 161
Applications of FRET in Microscopy	p. 164
Emerging Applications Including Novel FRET Probes	p. 169
Intramolecular FRET	p. 169
Intermolecular FRET	p. 171
Advanced FRET Methods	p. 171
References	p. 172
Monitoring Molecular Dynamics in Live Cells Using Fluorescence Photobleaching	p. 175
Introduction	p. 175
Photobleaching Theory	p. 176
Dynamics of Macromolecules	p. 177
Macromolecular Mobilities	p. 177
Fluorescence Recovery After Photobleaching	p. 179
Photobleaching Measurements with Confocal Microscope	p. 183
Photobleaching Applications	p. 187
Macromolecular Organization	p. 187
Monitoring Movement between Different Compartments	p. 190
Fluorescence Loss in Photobleaching	p. 191
Fluorescence Resonance Energy Transfer	p. 192
Conclusions	p. 194
References	p. 194
Time-Resolved Fluorescence in Microscopy	p. 195
Introduction	p. 195
Photophysics and Deactivation of Excited State	p. 195
Time-Resolved Fluorescence Measurements	p. 197
Deviations from Ideal Decay Behavior	p. 198
Time-Resolved Fluorescence Techniques	p. 200
Time-Resolved Fluorescence in Microscopy	p. 206
Factors That Influence Choice of TRFM Method	p. 213
Conclusions	p. 218
References	p. 218
Fluorescence Correlation Spectroscopy	p. 223
Introduction	p. 223
Optics of Fluorescence Correlation Spectroscopy	p. 227
Practical Aspects of FCS Experiments	p. 229
Quantitative Evaluation of FCS Measurements to Obtain Diffusion Constants and Concentration	p. 230
One-Focus FCS	p. 230
Two-Focus FCS	p. 237
Conclusion	p. 243
References	p. 243
Flow Cytometry	p. 245
What is Flow Cytometry?	p. 245
History of Flow Cytometry	p. 246
Flow Cytometry Fundamentals	p. 248
Sensitivity of Flow Cytometers	p. 251
Excitation Sources	p. 252
Laser Excitation	p. 252
Gas Laser Sources	p. 253
Diode Lasers	p. 254
Solid-State Lasers	p. 254
Signal Detection and Analysis	p. 255
Forward Scatter	p. 255
Side Scatter	p. 255
Fluorescence	p. 255
Issues in Flow Cytometry Using Fluorescence: Fluorescence Compensation	p. 256
Multiparameter Analysis	p. 258
Cell Sorting	p. 258
Applications of Flow Cytometry	p. 260
Calibration	p. 260
Use of Beads for Diagnostic Purposes	p. 261
Water Testing	p. 262
Milk Analysis	p. 262
Brewing and Wine Production	p. 263
Food Microbiology	p. 264
Quality Control in Microbiological Testing in Various Industries	p. 264
Cytometry: Where to Now?	p. 265
References	p. 266
Fluorescence in Diagnostic Imaging	p. 269
Introduction	p. 269
Principles of Fluorescence Applied to Medical Diagnosis	p. 269
Contrast Agents	p. 269
Delivery Issues	p. 273
Amplification Strategies	p. 273
Introduction to Imaging Techniques In Vivo	p. 275
Optical Fluorescence Imaging Techniques In Vivo	p. 275
Near-Infrared Fluorescence Imaging	p. 275
Fluorescence Reflective Imaging	p. 275
Fluorescence Molecular Tomography	p. 277
Superficial Confocal Imaging (Optical Coherence Tomography)	p. 278
Other Techniques	p. 280
Imaging of Whole-Body Biological Systems	p. 280
Superficial Tumors	p. 280
Subsurface Tumors and Vascularity	p. 281
Lymphoreticular System	p. 282
Bone	p. 283
Brain	p. 283
Future Directions	p. 283
References	p. 286
Fluorescence in Clinical Diagnosis	p. 289
Introduction	p. 289
Applications of Fluorescence in Clinical Biochemistry	p. 289
Advantages of Fluorescence Measurements	p. 289
Categories of Fluorescence Measurements	p. 290
Applications	p. 291
Fluorescence in Pathology and Cancer Diagnostics	p. 297
Fluorescent in Situ Hybridization	p. 297
Applications of Fluorescence in Clinical Cytology	p. 301
References	p. 304
Immunochemical Detection of Analytes by Using Fluorescence	p. 309
Introduction: Definition and General Principles of Immunoassay	p. 309
Immunoassay Types and Formats	p. 311
Competitive and Sandwich Immunoassays	p. 311
Homogeneous and Heterogeneous (Solid-Phase) Immunoassays	p. 311
Types of Labels Used in Immunoassays with Fluorescence Detection (Fluorophores, Enzymes with Fluorogenic Substrates)	p. 313
Types of Analytes	p. 313
Cations, Metal Ions, and Anions	p. 313
Small-Molecule Analytes (Pesticides, Toxins, and Biomarkers)	p. 314
Proteins	p. 314
Steady-State Fluorescence Immunoassays	p. 314
Intensity-Based (Quenching, Enhancement) Assays: Environmentally Sensitive Probes	p. 314
Energy Transfer Assays	p. 315
Chemiluminescence Assays: Enzyme Immunoassays Utilizing Fluorescent Substrates	p. 315
Polarization Assays	p. 317
Time-Resolved and Kinetic Approaches as Tools for Elimination of Fluorescence Background	p. 318
Kinetic Approach, Stopped-Flow Immunoassays, Time-Gated Detection, and Lifetime Assays	p. 318
Near-Infrared Fluorophores	p. 319
Recent Advances in Immunoassay Signal Enhancement and Throughput	p. 320
Metal-Particle-Enhanced Immunoassays: Nanoparticles as Labels	p. 320
Surface Plasmon-Coupled Emission-Based Immunoassays	p. 321
Assay Miniaturization (Arrays, Microchips)	p. 322
References	p. 322
Membrane Organization Astrid Magenau, Carles Rentero, and Katharina Gaus	p. 327
Concepts of Organization of Biological Membranes	p. 327
Bilayer	p. 327
Conceptual Models of Membrane Organization	p. 328
Fluorescence Methods to Study Membrane Organization	p. 330
Fluorescence Intensity Imaging and Membrane Probes	p. 330
Fluorescence Recovery After Photobleaching	p. 332
Fluorescence Correlation Spectroscopy and Image Correlation Spectroscopy	p. 333
Fluorescence Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy	p. 334
Total Internal Reflection Fluorescence Microscopy	p. 335
Single-Particle Tracking	p. 335
Model Membranes	p. 336
Membrane Organization in Cells	p. 340
Lipid Organization in Cell Membrane	p. 340
Protein-Lipid Interactions	p. 341
Protein-Protein Interactions	p. 345
References	p. 347
Probing Kinetics of Ion Pumps Via Voltage-Sensitive Fluorescent Dyes	p. 349
Introduction:Voltage-Dependent Physiological Processes, Ion Pumps, and Channels	p. 349
Voltage-Sensitive Dyes: Mechanisms, Response Times, and Their Relevance for Kinetic Studies	p. 351
Steady-State Ion Pump Activity	p. 355
Kinetics of Ion Pump Partial Reactions	p. 358
Future Directions	p. 361
References	p. 362
Index	p. 365
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Fluorescence Applications in Biotechnology and Life Sciences

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Fluorescence Applications in Biotechnology and Life Sciences

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