Standardization and Quality Assurance in Fluorescence Measurements II (eBook)

Series Editor 6
Volume Editor 6
Preface 7
Contents 9
Contributors 12
Part I Fluorescence Microscopy 16
Need for Standardization of Fluorescence Measurements from the InstrumentManufacturer’s View 17
1 Introduction 18
2 Standardization – but Which Parameters? 19
3 Overview on Calibration Methods for Confocal Microscopy 22
4 Use of a Thin Fluorescent Film Sample to Determine the Signal- to- Noise Ratio in a Confocal Microscope 23
5 Conclusion 32
References 38
Characterization and Calibration inWide Field and Sectioned Fluorescence Microscopy SIPcharts 39
1 Introduction 40
2 Image Calibration in Wide Field Fluorescence Microscopy 41
3 Characterization of Sectioning Fluorescence Microscopy (3D) with Thin Uniform Fluorescent Layers: Sectioned Imaging Property or SIPcharts 55
4 Conclusions 66
References 68
Quantitative Fluorescence Microscopy: Considerations and Controls 69
1 Introduction 70
2 The Intensity Dimension 72
3 The Spatial Dimension 86
4 The Temporal Dimension 98
5 Discussion 99
References 100
Comparability of Fluorescence Microscopy Data and Need for Instrument Characterization of Spectral Scanning Microscopes 103
1 Introduction 104
2 Instrument-Specific Parameters and Quantities Affecting Spectral Measurements in Microscopy 107
3 Comparability of Fluorescence Microscopy Data and Instrument Characterization 108
4 Conclusion, Future Requirements and Challenges 126
References 127
Fluorescence Lifetime Imaging Microscopy: Quality Assessment and Standards 131
1 Introduction 132
2 Förster Resonance Energy Transfer 134
3 Instrumentation and Techniques 135
4 Fluorophore Standards 139
5 System Calibration 141
6 Photon Efficiency and Photon Economy 143
7 Acquisition Speed 145
8 Data Quality Assessment and Analysis 146
9 Photobleaching 149
10 Fluorophore Saturation 150
11 Fluorescent Stainings 151
12 Conclusions 152
References 155
Part II Single Molecule Spectroscopy 157
State of the Art and Novel Trends in Fluorescence Correlation Spectroscopy 158
1 Introduction 159
2 General Principles of FCS 161
3 Experimental Aspects 178
4 Nonidealities and Artifacts 180
5 Evaluation of Experimental FCS Data 187
6 Some Application Aspects 192
7 Varieties of FCS and Related Techniques 197
8 Conclusions 202
References 202
Single Molecule Spectroscopy: Instrumentation and Multiparameter Detection 211
1 Introduction 211
2 Accessible Parameters 212
3 Instrumentation 215
4 Data Analysis in Multiparameter Measurements 218
References 223
Part III Fluorescence- Based Microarray Technology: Applications, Future Trends, and Need for Standardization 225
DNA Microarrays: Applications, Future Trends, and the Need for Standardization 226
1 Introduction 227
2 Microarray Technology 229
3 Microarray Applications 232
4 Future Trends 235
5 Need for Standardization 240
6 Conclusions 245
References 246
Comparability of Microarray Experiments from the Instrument and the Sample Site and Approaches Towards Standardization 249
1 Introduction 250
2 General Considerations About Microarray Experiments 251
3 Sources of Inconsistency in Microarray Measurements 257
4 Approaches Towards Standardization 258
5 Comparability of Microarray Experiments 263
6 Commercial DNA Microarray Platforms 267
7 Conclusion 270
References 270
Microarray Technology: Unresolved Issues and Future Challenges froma Regulatory Perspective 274
1 Introduction: The Apparent Lack of Reproducibility 275
2 Microarray Quality-Control Metrics and Thresholds: Assessing Data Quality from Various Perspectives 276
3 Consensus on Data-Analysis Methods: Adequate Evaluation Is Needed 279
4 The MicroArray Quality Control (MAQC) Project 283
5 Conclusions 288
References 290
Protein Arrays and Fluorescence Detection: Applications and Limitations 292
1 Introduction 293
2 Peptide and Protein Arrays: Composition, Production, and Processing 294
3 Applications of Protein Arrays 302
4 Quality Assurance of Array Data 308
References 311
Part IV Flow Cytometry 314
Flow Cytometry: Instrumentation, Applications, Future Trends and Limitations 315
1 Introduction 316
2 General Concept of Flow Cytometry with Brief History 317
3 Instrumentation Details 324
4 Optical Measurements 334
5 Limitations 342
6 Applications and Future Trends 345
References 347
Flow Cytometry Quality Assurance 351
1 Introduction 352
2 Precision 354
3 Sensitivity 363
4 Summary 376
References 376
Approaches to Quantitation in Flow Cytometry 379
1 Introduction 381
2 Fluorescence Signal 382
3 Conceptual Framework for Quantitation 383
4 Practical Assignment of MESF Values 385
5 Application of MESF 395
6 Conclusion 404
References 405
Part V Fluorescence Immunoassays 407
Immunoassays: Basic Concepts, Physical Chemistry and Validation 408
1 Introduction: Antibodies as an Analytical Tool 409
2 Antibodies and Recognition Reaction 410
3 Assay Formats – Assay Setups 414
4 Marker Systems and Conjugation 421
5 Assay Validation 425
6 Conclusions and Outlook 433
References 434
Time-Resolved Fluorometric Immunoassays Instrumentation, Applications, Unresolved Issues and Future Trends
1 Immunoassays as Analytical Methods in Diagnostics 436
2 Time-Resolved Fluorometry 438
3 Lanthanide Chelates and Assay Technologies 441
4 Problems of Standardization in Clinical Immunoassays 444
5 Immunofluorometric Applications 446
6 Future Trends and Challenges 449
References 451
Particle-Based Assays: Applications and Unresolved Issues 455
1 Introduction 456
2 Performance Criteria of Immunoassays 457
3 Why Use Particles? 459
4 General Aspects of Particle-Based Assays 461
5 Issues Concerning Specific Markers 465
6 Conclusion and Outlook 472
References 473
Advances in Fluorescence Enzyme Detection Methods 475
1 Introduction 476
2 Fluorigenic Substrates – Introduction 478
3 Fluorigenic Substrates Utilising Energy Transfer 482
4 Fluorescence Polarisation Enzyme Assays 484
5 Fluorigenic Substrates Yielding Insoluble Products 487
6 Intracellular Enzyme Assays 488
7 Multiplexed Enzyme Assays 488
8 Conclusions 490
References 490
Part VI Quantitative PCR 492
Quantitative Real-Time PCR: Fluorescent Probe Options and Issues 493
1 PCR 494
2 Real-Time Quantitative PCR (rt-Q-PCR) 495
3 Fluorescence Detection in Quantitative PCR: Nonspecific Detection 496
4 Fluorescence Detection in Quantitative PCR: Specific Detection 497
5 Conclusions 510
References 511
Part VII Fluorescence In Situ Hybridization and Immunohistochemistry 513
Cellular Bioimaging in Fluorescent Cancer Biomarker Evaluation: Validation, Technologies and Standards Development 514
1 Introduction: Validation, Heterogeneity and Cellular Cancer Biomarkers 515
2 Continuous and Discrete Values for Cancer Biomarkers, Cellular Heterogeneity and Measurement Issues 518
3 DNA and Antibody Probes: Fluorescence Reagents for Cancer Biomarker Quantification in Cells 520
4 Application to Quantitative Medical Histopathology 527
5 Significant Challenges to Implementation of Standards for Cancer Biomarkers 527
6 Physical Standards for Fluorescent Measurement of Cancer Biomarkers: Now or Later? 528
7 Summary 529
References 530
Part VIII Fluorescence Technologies in Biomedical Diagnostics 534
Fluorescence Techniques in Biomedical Diagnostics: Instrumentation, Analysis and Unresolved Issues 535
1 Overview 536
2 Methods, Instrumentation and Applications 536
3 Conclusions 547
References 548
In-vivo Fluorescence Imaging: Applications, Future Trends & Approaches to Standardization
1 Main Text 551
2 Diffuse Fluorescence Tomography 553
3 Relation of Inversion and Standardization 555
4 Current Diffusive Fluorescent Standards 558
5 Conclusion 561
References 561
Subject Index 563