Fluorescence Applications in Biotechnology and Life Sciences »

Fluorescence Applications in Biotechnology and the Life Sciences

Edited by

Ewa M. Goldys

A self-contained treatment of the latest fluorescence applications in biotechnology and the life sciences

Fluorescence Applications in Biotechnology and the Life Sciences is the first reference in this important subject area to focus specifically on the present applications of fluorescence in molecular and cellular dynamics, biological/medical imaging, proteomics, genomics, and flow cytometry. It is designed to raise awareness of the latest scientific approaches and technologies that may help resolve problems relevant for the industry and the community in areas such as public health, food safety, and environ-mental monitoring.

Following an introductory chapter on the basics of fluorescence, the book covers: labeling of cells with fluorescent dyes; genetically encoded fluorescent proteins; nanoparticle fluorescence probes; quantitative analysis of fluorescent images; spectral imaging and unmixing; correlation of light with electron microscopy; fluorescence resonance energy transfer and applications; monitoring molecular dynamics in live cells using fluorescence photo-bleaching; time-resolved fluorescence in microscopy; fluorescence correlation spectroscopy; flow cytometry; fluorescence in diagnostic imaging; fluorescence in clinical diagnoses; immunochemical detection of analytes by using fluorescence; membrane organization; and probing the kinetics of ion pumps via voltage-sensitive fluorescent dyes.

With its multidisciplinary approach and excellent balance of research and diagnostic topics, this book will appeal to postgraduate students and a broad range of scientists and researchers in biology, physics, chemistry, biotechnology, bioengineering, and medicine.

Preface xv

Acknowledgments xxi

About the Contributing Authors xxiii

1 Basics of Fluorescence Robert P. Learmonth Scott H. Kable Kenneth P. Ghiggino 1

1.1 Introduction 1

1.1.1 Properties of Light 2

1.1.2 States of Molecules 5

1.2 Absorption and Emission of Light 9

1.2.1 Interaction of Light with a Molecule 9

1.2.2 Absorption and Emission of Light Depicted on a Perrin-Jablonski Diagram 10

1.2.3 Multiphoton Excitation 11

1.3 Noradiative Decay Mechanisms 13

1.3.1 Vibrational Relaxation 13

1.3.2 Internal Conversion 14

1.3.3 Intersystem Crossing 14

1.4 Properties of Excited Molecules 16

1.4.1 Quantum Yield 16

1.4.2 Excited-State Lifetime 16

1.5 Spectroscopy and Fluorophores 16

1.5.1 Absorption, Excitation, and Emission Spectra 16

1.5.2 Basic Properties of Fluorophores 19

1.6 Environmental Sensitivity of Fluorophores 20

1.6.1 Quenching of Fluorescence 21

1.6.2 Photobleaching 21

1.6.3 Fluorescence Resonance Energy Transfer 22

1.6.4 Solvent Relaxation 22

1.7 Polarization of Fluorescence 24

1.8 Conclusion 26

References 26

2 Labeling of Cells with Fluorescent Dyes Ian S. Harper 27

2.1 Introduction 27

2.2 Fluorophores Selection 32

2.3 Loading and Labeling Live Cells 34

2.3.1 Loading AM Forms 35

2.3.2 Leakage and Hydrolysis of AM Esters 36

2.4 Fluorophores for Live Cell Imaging 36

2.4.1 Cell Viability 36

2.4.2 General Morphology 36

2.4.3 Specific Organelles 37

2.4.4 Ionic Environment 38

2.4.5 Tracers for Membranes and Cells 39

2.4.6 Fluorescent Proteins 40

References 41

3 Genetically Encoded Fluorescent Probes: Some Properties and Applications in Life Sciences Mark Prescott Anya Salih 47

3.1 Introduction47

3.2 Chromophore and its Formation 49

3.3 "Life and Death" of Fluorescent Protein 54

3.3.1 Oligomerization 57

3.3.2 Fusion Proteins 58

3.3.3 Photobleaching 58

3.3.4 Destabilization 59

3.4 Applications 60

3.5 Passive Applications 61

3.5.1 Photoamplifying PAFPs 62

3.5.2 Photoconverting PAFPs 63

3.5.3 Kindling PAFPs 64

3.5.4 Fluorescent Timers 64

3.6 Active Applications 65

3.6.1 Protein-Protein Interactions 65

3.6.2 Monitoring Enzyme Activity 66

3.6.3 Monitoring Small Molecules and Metabolites 66

3.6.4 Monitoring pH and Other Small Molecules 67

3.6.5 Monitoring Redox 67

3.7 Interactive Applications 67

3.8 Conclusions 68

References 69

4 Nanoparticle Fluorescence Probes Krystyna Drozdowicz-Tomsia Ewa M. Goldys 75

4.1 Introduction 75

4.2 Nanomaterials for Biological Applications 75

4.3 Inorganic Quantum Dots: Physics and Optical Properties 77

4.4 Synthesis of Monodisperse Colloidal Quantum Dots for Biolabeling Applications 81

4.4.1 Quantum Dot Synthesis 82

4.4.2 Shell Synthesis 84

4.4.3 Water Solubilization and Functionalization 85

4.4.4 Quantum Dot Bioconjugation 88

4.5 Quantum Dots as In Vitro Probes 89

4.6 Quantum Dots as In Vivo Probes 92

4.7 Cytotoxicity 94

4.8 Future Directions 94

References 95

5 Quantitative Analysis of Fluorescent Image: From Descriptive to Computational Microscopy Michal Marek Godlewaski Agnieszka Turowska Paulina Jedynak Daniel Martinez Puig Helena Nevalainen 99

5.1 Introduction 99

5.2 Advantages of Quantitative Analysis 100

5.3 Methods of Quantitative Analysis 101

5.4 Image Processing 113

References 115

6 Spectral Imaging and Unmixing Pascal Vallotton Aloke Phatak Mark Berman 117

6.1 Introduction 117

6.2 Instrumentation and Configurations for Hyperspectral Microscopes 119

6.3 Supervised and Unsupervised Unmixing 121

6.4 Supervised or Informed Unmixing 121

6.5 Unsupervised or Blind Unmixing 122

6.5.1 Iterated Constrained End-Members Algorithm 123

6.6 Spectral Clustering 124

6.7 Examples 125

6.7.1 Tablet Inspection 125

6.7.2 Fluorescent Microspheres 127

6.7.3 Induced Mouse Lung Tumor 131

6.8 Limitations of Spectral Imaging and Experimental Considerations 135

6.8.1 Fast Biological Processes 135

6.8.2 Calibration 136

6.8.3 Environmental Sensitivity 137

6.8.4 Background Fluorescence 137

6.9 Conclusions 137

References 138

7 Correlation of Light with Electron Microscopy: A Correlative Microscopy Platform Marko Nykäet;nen 141

7.1 Introduction 141

7.2 Overview of Techniques Used in Correlative Microscopy 142

7.3 Correlative Microscopy of Chemically Fixed, Immunolabeled Ultrathin Cryosections Employing Antibodies Coupled with Fluorescent Gold Conjugates 145

7.4 Special Techniques Used in Sample Preparation for Correlative Microscopy: High-Pressure Freezing, Freeze Fracturing, and Freeze Substitution 151

7.5 Correlative Microscopy of Fixed Tissue Specimens Using Freeze Substitution 153

7.6 Future Trends: Correlative Microscopy and High-Content Cellular Screening 154

7.7 Future Trends: Correlative Microscopy of Fluorescent Images Acquired by Confocal Laser Scanning Microscopy 154

References 155

8 Fluorescence Resonance Energy Transfer and Applications Andrew Clayton 157

8.1 What is FRET? 157

8.1.1 Theory of FRET 157

8.2 Why FRET Can Be Useful 159

8.3 How FRET Can Be Measured 160

8.3.1 Applications of FRET in Spectroscopy 161

8.3. Applications of FRET in Microscopy 164

8.4 Emerging Applications Including Novel FRET Probes 169

8.4.1 Intramolecular FRET 169

8.4.2 Intermolecular FRET 171

8.5 Advanced FRET Methods 171

References 172

9 Monitoring Molecular Dynamics in Live Cells Using Fluorescence Photobleaching Nectarios Klonis Leann Tilley 175

9.1 Introduction 175

9.2 Photobleaching Theory 176

9.3 Dynamics of Macromolecules 177

9.3.1 Macromolecular Mobilities 177

9.3.2 Fluorescence Recovery After Photobleaching 179

9.4 Photobleaching Measurements with Confocal Microscope 183

9.5 Photobleaching Applications 187

9.5.1 Macromolecular Organization 187

9.5.2 Monitoring Movement between Different Compartments 190

9.5.3 Fluorescence Loss in Photobleaching 191

9.5.4 Fluorescence Resonance Energy Transfer 192

9.6 Conclusions 194

References 194

10 Time-Resolved Fluorescence in Microscopy Trevor A. Smith Craig N. Lincoln Damian K. Bird 195

10.1 Introduction 195

10.2 Photophysics and Deactivation of Excited State 195

10.3 Time-Resolved Fluorescence Measurements 197

10.3.1 Deviations from Ideal Decay Behavior 198

10.3.2 Time-Resolved Fluorescence Techniques 200

10.4 Time-Resolved Fluorescence in Microscopy 206

10.4.1 Factors That Influence Choice of TRFM Method 213

10.5 Conclusions 218

References 218

11 Fluorescence Correlation Spectroscopy Thomas Dertinger Iris von der Hocht Anastasia Loman Ingo Gregor Jöet;rg Enderlein 223

11.1 Introduction 223

11.2 Optics of Fluorescence Correlation Spectroscopy 227

11.3 Practical Aspects of FCS Experiments 229

11.4 Quantitative Evaluation of FCS Measurements to Obtain Diffusion Constants and Concentration 230

11.4.1 One-Focus FCS 230

11.4.2 Two-Focus FCS 237

11.5 Conclusion 243

References 243

12 Flow Cytometry Russell E. Connally Graham Vesey Charlotte Morgan 245

12.1 What is Flow Cytometry? 245

12.1.1 History of Flow Cytometry 246

12.1.2 Flow Cytometry Fundamentals 248

12.1.3 Sensitivity of Flow Cytometers 251

12.2 Excitation Sources 252

12.2.1 Laser Excitation 252

12.2.2 Gas Laser Sources 253

12.2.3 Diode Lasers 254

12.2.4 Solid-State Lasers 254

12.3 Signal Detection and Analysis 255

12.3.1 Forward Scatter 255

12.3.2 Side Scatter 255

12.3.3 Fluorescence 255

12.3.4 Issues in Flow Cytometry Using Fluorescence: Fluorescence Compensation 256

12.3.5 Multiparameter Analysis 258

12.4 Cell Sorting 258

12.5 Applications of Flow Cytometry 260

12.5.1 Calibration 260

12.5.2 Use of Beads for Diagnostic Purposes 261

12.5.3 Water Testing 262

12.5.4 Milk Analysis 262

12.5.5 Brewing and Wine Production 263

12.5.6 Food Microbiology 264

12.5.7 Quality Control in Microbiological Testing in Various Industries 264

12.5.8 Cytometry: Where to Now? 265

References 266

13 Fluorescence in Diagnostic Imaging Morry Silberstein 269

13.1 Introduction 269

13.2 Principles of Fluorescence Applied to Medical Diagnosis 269

13.2.1 Contrast Agents 269

13.2.2 Delivery Issues 273

13.2.3 Amplification Strategies 273

13.2.4 Introduction to Imaging Techniques In Vivo 275

13.3 Optical Fluorescence Imaging Techniques In Vivo 275

13.3.1 Near-Infrared Fluorescence Imaging 275

13.3.2 Fluorescence Reflective Imaging 275

13.3.3 Fluorescence Molecular Tomography 277

13.3.4 Superficial Confocal Imaging (Optical Coherence Tomography) 278

13.3.5 Other Techniques 280

13.4 Imaging of Whole-Body Biological Systems 280

13.4.1 Superficial Tumors 280

13.4.2 Subsurface Tumors and Vascularity 281

13.4.3 Lymphoreticular System 282

13.4.4 Bone 283

13.4.5 Brain 283

13.5 Future Directions 283

References 286

14 Fluorescence in Clinical Diagnosis Wouter Kalle Phillip Bwititi Todd Walker 289

14.1 Introduction 289

14.2 Applications of Fluorescence in Clinical Biochemistry 289

14.2.1 Advantages of Fluorescence Measurements 289

14.2.2 Categories of Fluorescence Measurements 290

14.2.3 Applications 291

14.3 Fluorescence in Pathology and Cancer Diagnostics 297

14.3.1 Fluorescent in Situ Hybridization 297

14.3.2 Applications of Fluorescence in Clinical Cytology 301

References 304

15 Immunochemical Detection of Analytes by Using Fluorescence Evgenia G. Matveeva Ignacy Gryczynski Zygmunt Gryczynski Ewa M. Goldys 309

15.1 Introduction: Definition and General Principles of Immunoassay 309

15.2 Immunoassay Types and Formats 311

15.2.1 Competitive and Sandwich Immunoassays 311

15.2.2 Homogeneous and Heterogeneous (Solid-Phase) Immunoassays 311

15.2.3 Types of Labels Used in Immunoassays with Fluorescence Detection (Fluorophores, Enzymes with Fluorogenic Substrates) 313

15.3 Types of Analytes 313

15.3.1 Cations, Metal Ions, and Anions 313

15.3.2 Small-Molecule Analytes (Pesticides, Toxins, and Biomarkers) 314

15.3.3 Proteins 314

15.4 Steady-State Fluorescence Immunoassays 314

15.4.1 Intensity-Based (Quenching, Enhancement) Assays: Environmentally Sensitive Probes 314

15.4.2 Energy Transfer Assays 315

15.4.3 Chemiluminescence Assays: Enzyme Immunoassays Utilizing Fluorescent Substrates 315

15.4.4 Polarization Assays 317

15.5 Time-Resolved and Kinetic Approaches as Tools for Elimination of Fluorescence Background 318

15.5.1 Kinetic Approach, Stopped-Flow Immunoassays, Time-Gated Detection, and Lifetime Assays 318

15.5.2 Near-Infrared Fluorophores 319

15.6 Recent Advances in Immunoassay Signal Enhancement and Throughput 320

15.6.1 Metal-Particle-Enhanced Immunoassays: Nanoparticles as Labels 320

15.6.2 Surface Plasmon-Coupled Emission-Based Immunoassays 321

15.6.3 Assay Miniaturization (Arrays, Microchips) 322

References 322

16 Membrane Organization Astrid Magenau, Carles Rentero, and Katharina Gaus 327

16.1 Concepts of Organization of Biological Membranes 327

16.1.1 Bilayer 327

16.1.2 Conceptual Models of Membrane Organization 328

16.2 Fluorescence Methods to Study Membrane Organization 330

16.2.1 Fluorescence Intensity Imaging and Membrane Probes 330

16.2.2 Fluorescence Recovery After Photobleaching 332

16.2.3 Fluorescence Correlation Spectroscopy and Image Correlation Spectroscopy 333

16.2.4 Fluorescence Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy 334

16.2.5 Total Internal Reflection Fluorescence Microscopy 335

16.2.6 Single-Particle Tracking 335

16.3 Model Membranes 336

16.4 Membrane Organization in Cells 340

16.4.1 Lipid Organization in Cell Membrane 340

16.4.2 Protein-Lipid Interactions 341

16.4.3 Protein-Protein Interactions 345

References 347

17 Probing Kinetics of Ion Pumps Via Voltage-Sensitive Fluorescent Dyes Ronald J. Clarke 349

17.1 Introduction:Voltage-Dependent Physiological Processes, Ion Pumps, and Channels 349

17.2 Voltage-Sensitive Dyes: Mechanisms, Response Times, and Their Relevance for Kinetic Studies 351

17.3 Steady-State Ion Pump Activity 355

17.4 Kinetics of Ion Pump Partial Reactions 358

17.5 Future Directions 361

References 362

Index 365