Imaging Cellular and Molecular Biological Functions (eBook)

Preface 5
Contents 8
Contributors 16
Considerations for Routine Imaging 21
Entering the Portal: Understanding the Digital Image Recorded Through a Microscope 22
1.1 Introduction 22
1.2 Historical Perspective 23
1.3 Digital Image Acquisition: Analog to Digital Conversion 23
1.4 Spatial Resolution in Digital Images 25
1.5 The Contrast Transfer Function 27
1.6 Image Brightness and Bit Depth 29
1.7 Image Histograms 30
1.8 Fundamental Properties of CCD Cameras 31
1.9 CCD Enhancing Technologies 35
1.10 CCD Performance Measures 36
1.11 Multidimensional Imaging 40
1.12 The Point-Spread Function 43
1.13 Digital Image Display and Storage 47
1.14 Imaging Modes in Optical Microscopy 48
1.15 Summary 58
1.16 Internet Resources 60
References 60
Quantitative Biological Image Analysis 63
2.1 Introduction 63
2.2 Definitions and Perspectives 64
2.3 Image Preprocessing 66
2.4 Advanced Processing for Image Analysis 75
2.5 Higher-Dimensional Data Visualization 81
2.6 Software Tools and Development 84
References 86
The Open Microscopy Environment: A Collaborative Data Modeling and Software Development Project for Biological Image Informatics 89
3.1 Introduction 89
3.2 OME Specifications and File Formats 92
3.3 OME Data Management and Analysis Software 95
3.4 Conclusions and Future Directions 108
References 108
Design and Function of a Light-Microscopy Facility 111
4.1 Introduction 111
4.2 Users 113
4.3 Staff 114
4.4 Equipment 116
4.5 Organization 121
4.6 Summary 130
References 131
Advanced Methods and Concepts 132
Quantitative Colocalisation Imaging: Concepts, Measurements, and Pitfalls 133
5.1 Introduction 133
5.2 Quantifying Colocalisation 153
5.3 Conclusions 166
References 167
Quantitative FRET Microscopy of Live Cells 172
6.1 Introduction 172
6.2 Introductory Physics of FRET 173
6.3 Manifestations of FRET in Fluorescence Signals 175
6.4 Molecular Interaction Mechanisms That Can Be Observed by FRET 178
6.5 Measuring Fluorescence Signals in the Microscope 180
6.6 Methods for FRET Microscopy 182
6.7 Fluorescence Lifetime Imaging Microscopy for FRET 190
6.8 Data Display and Interpretation 191
6.9 FRET-Based Biosensors 192
6.10 FRET Microscopy for Analyzing Interaction Networks in Live Cells 193
6.11 Conclusion 195
References 195
Fluorescence Photobleaching and Fluorescence Correlation Spectroscopy: Two Complementary Technologies To Study Molecular Dynamics in Living Cells 197
7.1 Introduction 197
7.2 Fundamentals 203
7.3 How To Perform a FRAP Experiment 210
7.4 How To Perform an FCS Experiment 219
7.5 How To Perform a CP Experiment 231
7.6 Quantitative Treatment 235
7.7 Conclusion 241
References 241
Single Fluorescent Molecule Tracking in Live Cells 248
8.1 Introduction 248
8.2 Tracking of Single Chromosomal Loci 249
8.3 Single-Molecule Tracking of mRNA 260
8.4 Single-Particle Tracking for Membrane Proteins 266
8.5 Tracking Analysis and Image Processing of Data from Particle Tracking in Living Cells 271
8.6 Conclusion 271
8.7 Protocols for Laboratory Use 272
References 274
From Live-Cell Microscopy to Molecular Mechanisms: Deciphering the Functions of Kinetochore Proteins 277
9.1 Introduction 277
9.2 Biological Problem: Deciphering the Functions of Kinetochore Proteins 280
9.3 Experimental Design 281
9.4 Extraction of Dynamics from Images 285
9.5 Characterization of Dynamics 288
9.6 Quantitative Genetics of the Yeast Kinetochore 294
9.7 Conclusion 296
References 296
Cutting Edge Applications & Utilities
Towards Imaging the Dynamics of Protein Signalling 299
10.1 Spatiotemporal Aspects of Protein Signalling Dynamics 299
10.2 How to Be Fast While Maintaining the Resolution 300
10.3 How To Make Proteins Visible 309
10.4 Concepts To Image Protein Dynamics 313
10.5 Concepts To Image Protein–Protein Interactions 315
10.6 Concepts To Image Biochemistry with Fluorescent Proteins 319
References 321
New Technologies for Imaging and Analysis of Individual Microbial Cells 323
11.1 Introduction 323
11.2 Live-Cell Imaging 324
11.3 Imaging Infection 325
11.4 Imaging Single Molecules (Within Single Cells) 328
11.5 Measuring Discrete Cell Properties and Processes 329
11.6 “Wetware” 331
11.7 Hardware and Applications 333
11.8 Fluorescence Correlation Spectroscopy 336
11.9 A Picture is Worth a Thousand Dots – New Developments in Flow Cytometry 340
11.10 Strength in Numbers – Highly Parallel Analysis Using Cellular Arrays 344
11.11 Nontactile Manipulation of Individual Cells and “ Wall- less Test Tubes” 345
11.12 Conclusions 347
References 348
Imaging Parasites in Vivo 354
12.1 Introduction 354
12.2 The Life Cycle of Malaria Parasites 355
12.3 A Very Brief History of Light Microscopy and Malaria Parasites 357
12.4 In Vivo Imaging of Luminescent Parasites 358
12.5 In Vivo Imaging of Fluorescent Parasites 359
12.6 Imaging Malaria Parasites in the Mosquito 360
12.7 Imaging Malaria Parasites in the Mammalian Host 363
12.8 Towards Molecular Imaging in Vivo 367
12.9 A Look at Other Parasites 368
12.10 Conclusion 369
References 369
Computer-Assisted Systems for Dynamic 3D Reconstruction and Motion Analysis of Living Cells 374
13.1 Introduction 374
13.2 Approaches to 3D Reconstruction and Motion Analysis 375
13.3 Obtaining Optical Sections for 3D Reconstruction 377
13.4 Outlining 377
13.5 Reconstructing 3D Faceted Images and Internal Architecture 382
13.6 Quantitative Analyses of Behavior 382
13.7 3D-DIASemb 384
13.8 Resolving Filopodia 386
13.9 The Combined Use of LSCM and 3D-DIAS 389
13.10 Reasons for 3D Dynamic Image Reconstruction Analysis 390
References 391
High-Throughput/High-Content Automated Image Acquisition and Analysis 394
14.1 The Driving Forces for High-Throughput/High-Content Automated Imaging 394
14.2 Confocal Imaging in High Throughput – The Principles Available 395
14.3 Resolution and Sensitivity 398
14.4 Measurements 401
14.5 Where Is the Signal and How To Focus? 402
14.6 Plates and Lenses 403
14.7 Image Analysis 404
14.8 Throughput: How to Acquire and Analyze Data Rapidly 408
14.9 Screening Examples 410
References 413
Cognition Network Technology – A Novel Multimodal Image Analysis Technique for Automatic Identification and Quantification of Biological Image Contents 415
15.1 Introduction 415
15.2 Cognition Network Technology and Cognition Network Language 417
15.3 Discussion 429
References 429
High-Content Phenotypic Cell-Based Assays 431
16.1 A New Tool for Biological Research and Drug Discovery 431
16.2 What Is High-Content Screening and How Can Biologists Use It? 432
16.3 Assay Design: First Think, Then Act 433
16.4 Assay Optimization 434
16.5 Cell Culture 434
16.6 Cell Vessels 436
16.7 Cellular Imaging 436
16.8 Autofluorescence 438
16.9 Image Analysis 439
16.10 Transfection Optimization for RNAi-Based Assays 439
16.11 Escapers and Silencing Efficiency 440
16.12 Toxicity 443
16.13 Off-Target or Unspecific Reactions 444
16.14 Assay Quality 445
16.15 Assay Validation 446
16.16 Conclusion and Outlook 448
References 448
Index 451