Front Cover 1
Quantitative Imaging in Cell Biology 4
Copyright 5
Contents 6
Contributors 14
Preface 20
Chapter 1: Concepts in quantitative fluorescence microscopy 22
1.1. Accurate and Precise Quantitation 23
1.2. Signal, Background, and Noise 24
1.3. Optical Resolution: The Point Spread Function 28
1.4. Choice of Imaging Modality 28
1.5. Sampling: Spatial and Temporal 29
1.5.1. 2D sampling 31
1.5.2. 3D sampling 32
1.5.3. Temporal sampling 33
1.6. Postacquisition Corrections 33
1.6.1. Background subtraction 33
1.6.2. Flat-field correction 34
1.6.3. Photobleaching 35
1.6.4. Storing and processing images for quantitation 35
1.7. Making Compromises 36
1.8. Communicating Your Results 37
Acknowledgment 37
References 37
Chapter 2: Practical considerations of objective lenses for application in cell biology 40
Introduction 41
2.1. Optical Aberrations 41
2.1.1. On-axis aberrations 43
2.1.2. Off-axis aberrations 43
2.2. Types of Objective Lenses 43
2.2.1. Optical corrections 44
2.2.2. Numerical aperture 46
2.3. Objective Lens Nomenclature 46
2.4. Optical Transmission and Image Intensity 46
2.5. Coverslips, Immersion Media, and Induced Aberration 48
2.5.1. Optical path length 48
2.5.2. Correction collars 49
2.5.3. Cover glass 50
2.5.4. Immersion media 51
2.6. Considerations for Specialized Techniques 52
2.7. Care and Cleaning of Optics 53
Conclusions 55
References 55
Chapter 3: Assessing camera performance for quantitative microscopy 56
3.1. Introduction to Digital Cameras for Quantitative Fluorescence Microscopy 57
3.2. Camera Parameters 58
3.2.1. Quantum efficiency 58
3.2.2. Noise 58
3.2.3. Poisson noise 58
3.2.4. Camera noise 60
3.2.5. Fixed-Pattern noise 60
3.2.6. Digitization, bit depth, and dynamic range 61
3.2.7. Amplification 62
3.2.8. sCMOS considerations 64
3.3. Testing Camera Performance: The Photon Transfer Curve 65
3.3.1. Photon transfer theory 65
3.3.2. PTC collection protocol 67
References 73
Chapter 4: A Practical guide to microscope care and maintenance 76
Introduction 77
4.1. Cleaning 79
4.1.1. Before cleaning 79
4.1.2. Objectives 80
4.1.2.1. Proper use of objective lenses 80
4.1.2.2. Objective lens inspection and cleaning 81
4.1.2.3. Temperature 83
4.1.3. Fluorescence filters 83
4.1.3.1. Excitation and emission filters 84
4.1.3.2. Mirrors 85
4.1.4. Camera 85
4.1.5. The dust is still there! 86
4.2. Maintenance and Testing 87
4.2.1. Computer maintenance 87
4.2.2. Check the transmitted light pathway 87
4.2.3. Measure intensity of fluorescence light sources 88
4.2.4. Flatness of fluorescence illumination 92
4.2.5. Color registration 92
4.2.6. Vibration 93
4.2.7. Measure the point spread function 94
4.2.8. Test performance of motorized components and software 94
4.3. Considerations for New System Installation 95
Acknowledgments 96
References 96
Chapter 5: Fluorescence live cell imaging 98
5.1. Fluorescence Microscopy Basics 99
5.2. The Live Cell Imaging Microscope 100
5.3. Microscope Environmental Control 104
5.3.1. Temperature 104
5.3.2. Media composition and pH 105
5.3.3. Imaging chambers 106
5.4. Fluorescent Proteins 108
5.4.1. Protocol for analyzing FP photobleaching 111
5.5. Other Fluorescent Probes 113
Conclusion 114
Acknowledgments 114
References 114
Chapter 6: Fluorescent proteins for quantitative microscopy: Important properties and practical evaluation 116
6.1. Optical and Physical Properties Important for Quantitative Imaging 117
6.1.1. Color and brightness 117
6.1.2. Photostability 119
6.1.3. Other properties 119
6.2. Physical Basis for Fluorescent Protein Properties 120
6.2.1. Determinants of wavelength 120
6.2.2. Determinants of brightness 121
6.3. The Complexities of Photostability 122
6.3.1. Multiple photobleaching pathways 123
6.3.2. Photobleaching behaviors 123
6.3.3. Reporting standards for FP photostability 127
6.4. Evaluation of Fluorescent Protein Performance in Vivo 127
6.4.1. Cell-line-specific photostability and contrast evaluation 128
Protocol 128
6.4.2. Fusion Protein-specific FP evaluation 129
Conclusion 129
References 130
Chapter 7: Quantitative confocal microscopy: Beyond a pretty picture 134
7.1. The Classic Confocal: Blocking Out the Blur 135
7.2. You Call that quantitative? 139
7.2.1. Quantitative imaging toolkit 139
7.2.2. Localization and morphology 140
7.2.3. Quantifying intensity 141
7.2.3.1. Aspects of the microscope 141
7.2.3.2. Aspects of the sample 142
7.2.3.2.1. Mounting media 142
7.2.3.2.2. Coverslips 144
7.2.3.2.3. Sample labeling 144
7.3. Interaction and Dynamics 144
7.3.1. Cross talk 144
7.3.2. Time-lapse imaging 145
7.3.3. Spectral imaging 146
7.4. Controls: Who Needs Them? 146
7.4.1. Unlabeled sample 146
7.4.2. Nonspecific binding controls 146
7.4.3. Antibody titration curves 146
7.4.4. Isotype controls 147
7.4.5. Blind imaging 148
7.4.6. Fluorescent proteins 148
7.4.7. Flat-field images 148
7.4.8. Biological control samples 148
7.5. Protocols 148
7.5.1. Protocol 1: Measuring instrument PSF (Resolution and objective Lens Quality) 148
Imaging 148
7.5.2. Protocol 2: Testing short-term and long-term laser power stability 150
Slide 150
Data Collection 150
Data Analysis 150
7.5.3. Protocol 3: Correct nonuniform field illumination 150
Slide 150
Data Collection 150
Data Analysis 150
7.5.4. Protocol 4: Coregistration of TetraSpeck beads 151
Slide 151
Data Collection 151
Data Analysis 151
7.5.5. Protocol 5: Spectral accuracy 151
Slides 151
Data Collection 151
Data Analysis 152
7.5.6. Protocol 6: Spectral Unmixing algorithm accuracy 152
Slide 152
7.5.6.1. Channel (Multi-PMT) method 152
7.5.6.2. Separation (Unmixing) 153
7.5.6.3. Spectral detection method 153
Conclusions 154
References 154
Chapter 8: Assessing and benchmarking multiphoton microscopes for biologists 156
Introduction: Practical Quantitative 2P Benchmarking 157
8.1. Part I: Benchmarking Inputs 157
8.1.1. Laser power at the sample 158
8.1.2. Photomultiplier settings 159
8.1.2.1. Method 1—Fixed PMT voltage 160
8.1.2.2. Method 2—PMT voltage range 161
8.1.3. Standard samples 161
8.1.3.1. A standard three-dimensional sample set with variable dispersive properties 162
8.1.3.1.1. Support protocol: Preparation of PSF beads in a dispersive or nondispersive support 162
8.1.3.2. Standard biological samples 164
8.1.4. Sample-Driven parameters: How fast/How long 164
8.2. Part II: Benchmarking Outputs 165
8.2.1. The point spread function 165
8.2.2. SNR and total intensity 167
8.2.3. Maximal depth of acquisition 169
8.3. Troubleshooting/Optimizing 171
8.4. A Recipe for Purchasing Decisions 171
Conclusion 172
Acknowledgments 172
References 172
Chapter 9: Spinning-disk confocal microscopy: present technology and future trends 174
9.1. Principle of Operation 174
9.2. Strengths and Weaknesses 176
9.3. Improvements in Light Sources 178
9.4. Improvements in Illumination 178
9.5. Improvements in Optical Sectioning and FOV 183
9.6. New Detectors 187
9.7. A Look into the Future 188
References 192
Chapter 10: Quantitative deconvolution microscopy 198
Introduction 199
10.1. The Point-spread Function 201
10.2. Deconvolution Microscopy 203
10.2.1. Deblurring 204
10.2.2. Image restoration 205
10.2.3. Fourier transforms 205
10.2.4. Iterative methods 206
10.2.5. The importance of image quality 207
10.2.5.1. Factors that affect image restoration 207
10.3. Results 208
10.3.1. Assessing linearity 210
10.3.2. Applications of deconvolution microscopy 211
Conclusion 212
References 212
Chapter 11: Light sheet microscopy 214
Introduction 215
11.1. Principle of Light Sheet Microscopy 216
11.1.1. Light sheet illumination 216
11.1.2. Wide-Field detection 217
11.1.3. Large samples 219
11.2. Implementations of Light Sheet Microscopy 219
11.2.1. Light sheet properties 219
11.2.2. How to generate a light sheet 220
11.2.3. Vertical versus horizontal arrangements 222
11.2.4. Microscope built around the sample 222
11.2.5. Objective lenses 224
11.3. Mounting a Specimen for Light Sheet Microscopy 224
11.3.1. Solid gel cylinder 224
11.3.2. Tube embedding 226
11.4. Acquiring Data 226
11.4.1. Orienting the specimen 228
11.4.2. Light sheet alignment 228
11.4.2.1. Adjusting the light sheet height 228
11.4.2.2. Adjusting the light sheet thickness 229
11.4.2.3. Correct position of the beam waist 229
11.4.2.4. Moving the sheet in focus 230
11.4.2.5. Eliminating tilt 230
11.4.3. Choosing the right imaging parameters 230
11.5. Handling of Light Sheet Microscopy Data 231
11.5.1. Coping with high-Speed and large data 231
11.5.2. Image enhancements 232
11.5.3. Multiview fusion 232
11.5.4. Image Analysis 233
References 233
Chapter 12: DNA curtains: Novel tools for imaging protein–nucleic acid interactions at the single-molecule level 238
Introduction 239
12.1. Overview of TIRFM 240
12.2. Flow Cell Assembly 241
12.3. Importance of the Lipid Bilayer 242
12.4. Barriers to Lipid Diffusion 243
12.5. Different Types of DNA Curtains 244
12.5.1. Single-Tethered curtains 244
12.5.2. Double-Tethered DNA curtains 244
12.5.3. Parallel array of double-Tethered isolated patterns and crisscrossed DNA curtains 246
12.5.4. ssDNA curtains 246
12.6. Using DNA Curtains to Visualize Protein-DNA Interactions 247
12.6.1. Binding site preferences 247
12.6.2. Target search mechanisms 248
12.6.3. Protein–Protein colocalization 252
12.6.4. ATP hydrolysis-Driven DNA translocation 252
12.6.5. Beyond nucleic acids 252
12.7. Future Perspectives 253
Acknowledgments 253
References 253
Chapter 13: Nanoscale cellular imaging with scanning angle interference microscopy 256
Introduction 257
Superresolution optical imaging 257
Theory of SAIM 259
13.1. Experimental Methods and Instrumentation 262
13.1.1. Microscope and instrumentation 262
13.1.2. Preparation of reflective substrates 263
13.1.3. Selection of fluorescent probes 263
13.1.4. Cell culture and transfection 264
13.1.5. Immunolabeling of samples 265
13.1.6. Microscope calibration and configuration 267
13.1.7. Image acquisition 268
13.2. Image Analysis and Reconstruction 271
Conclusion 271
Acknowledgments 272
References 272
Chapter 14: Localization microscopy in yeast 274
Introduction 275
14.1. Preparing the Yeast Strain 277
14.2. Considerations for the Choice of a Labeling Strategy 278
14.3. Preparing the Sample 281
14.3.1. Immobilizing and fixing the yeast cells on coverslips 281
14.3.1.1. Materials and reagents 281
14.3.1.1.1. ConA-coated coverslips 281
14.3.1.1.2. ConA-cross-linked coverslips 282
14.3.1.2. Procedure 282
14.3.2. Labeling with organic dyes 283
14.3.2.1. Materials and reagents 283
14.3.2.1.1. Labeling of anti-GFP nanobodies with Alexa Fluor 647 284
14.3.2.1.2. Labeling of ConA with CF™ 680 284
14.3.2.2. Procedure: Nanobody or SNAP-tag staining 285
14.3.2.3. Procedure: ConA staining 285
14.4. Image Acquisition 285
14.4.1. Materials 285
14.5. Results 286
Summary 288
Acknowledgments 290
References 290
Chapter 15: Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM 294
Introduction 295
15.1. Principles 296
15.1.1. 2D superresolution microscopy by Photoactivated Localization Microscopy (PALM) 296
15.1.2. 3D superresolution by iPALM 296
15.2. Methods 298
15.2.1. Instrumentation for PALM 298
15.2.2. Fluorophore choice and sample preparation for PALM and iPALM 300
15.2.3. Fiducial-Based alignment: Drift correction and multichannel registration 304
15.2.4. Implementation of iPALM 307
15.2.5. Extending iPALM imaging depth with astigmatic defocusing 311
15.3. Future Directions 311
Acknowledgments 312
References 313
Chapter 16: Seeing more with structured illumination microscopy 316
Introduction 317
16.1. Theory of Structured Illumination 318
16.1.1. 2D image formation 318
16.1.2. Structured illumination 319
16.1.3. SIM combined with total internal reflection fluorescence Microscopy 322
16.2. 3D SIM 323
16.2.1. 3D image formation 323
16.2.2. 3D SIM theory 324
16.2.3. Practical implementations of 3D SIM 326
16.3. SIM Imaging Examples 328
16.3.1. TIRF SIM application 328
16.3.2. 3D SIM applications 328
16.4. Practical Considerations and Potential Pitfalls 331
16.5. Discussion 332
References 333
Chapter 17: Structured illumination superresolution imaging of the cytoskeleton 336
Introduction 337
Superresolution microscopy 337
SIM for imaging of the cytoskeleton 337
17.1. Instrumentation for SIM Imaging 337
17.1.1. Illumination pattern 338
17.1.2. Objective and camera 338
17.1.3. Reconstruction and judging of SIM images 342
17.2. Sample Preparation 343
17.2.1. Materials 343
Coverslip dishes 343
Fixative 343
Primary antibodies 343
Secondary antibodies 343
Fluorescent dyes 343
Mounting and index matching 343
Bead samples 343
17.2.2. Choice of fluorophore and staining 343
Indirect immunofluorescence 344
Genetically encoded fluorescence (fusion proteins with fluorescent proteins) 344
17.2.3. Fixation 344
17.2.4. Index matching and embedding of sample 344
17.3. Minimizing Spherical Aberration 345
17.3.1. What is spherical aberration and when does it occur 345
17.3.2. Steps to minimize spherical aberration on the microscope 346
Prepare bead sample 346
Measure point spread function 347
Qualitative assessment of PSF 347
Quantitative assessment 347
17.4. Multichannel SIM 348
17.4.1. Setting up multicolor SIM 348
17.4.2. Correction for chromatic shift 349
17.4.3. Colocalization 349
17.4.4. Notes on quantitative analysis of intensity distribution 349
17.5. Live Imaging with SIM 351
Acknowledgments 352
References 352
Chapter 18: Analysis of focal adhesion turnover: A quantitative live-cell imaging example 356
Introduction to Focal Adhesion Dynamics 356
18.1. FA Turnover Analysis 358
18.1.1. Sample preparation 358
18.1.2. Imaging 360
18.1.3. Image analysis 361
18.1.4. Data analysis 362
Acknowledgments 367
References 367
Chapter 19: Determining absolute protein numbers by quantitative fluorescence microscopy 368
Introduction 369
19.1. Methods for Counting Molecules 369
19.1.1. Imaging and measurement considerations 369
19.1.2. Fluorescence correlation spectroscopy 370
19.1.3. Stepwise photobleaching 372
19.1.4. Ratiometric comparison of fluorescence intensity to known standards 373
19.1.5. Fluorescence standards 375
19.2. Protocol for Counting Molecules by Ratiometric Comparison of Fluorescence Intensity 377
19.2.1. Minimizing instrument error 377
19.2.2. Measuring instrument variation 378
19.2.3. Budding yeast imaging protocol 379
19.2.4. Measuring Background-Subtracted, integrated intensity 379
19.2.5. Depth correction 380
19.2.6. Calculating photobleaching correction factor 381
19.2.7. Gaussian fitting and ratiometric comparison to determine Protein count 381
Conclusions 382
References 382
Chapter 20: High-Resolution Traction Force Microscopy 388
Introduction 389
Basic principle of high-Resolution Traction Force Microscopy (TFM) 391
Principles of traction reconstruction 393
Overview of methods for reconstruction of traction Forces 394
High resolution and regularization 395
20.1. Materials 395
20.1.1. Instrumentation for high-Resolution TFM 396
20.1.2. Polyacrylamide substrates with two colors of fiducial markers 398
20.1.2.1. Suggested equipment and materials 398
20.1.2.2. Protocol 399
20.1.3. Functionalization of polyacrylamide substrates with ECM proteins 401
20.1.3.1. Suggested equipment and materials 401
20.1.3.2. Protocol 402
20.2. Methods 402
20.2.1. Cell culture and preparation of samples for High-Resolution TFM 402
20.2.1.1. Suggested equipment and materials 403
20.2.1.2. Protocol 403
20.2.2. Setting up a perfusion chamber for TFM and acquiring TFM images 404
20.2.2.1. Suggested equipment and materials 404
20.2.2.2. Protocol 404
20.2.3. Quantifying deformation of the elastic substrate 406
20.2.4. Calculation of traction Forces with regularized Fourier–Transform Traction cytometry 408
20.2.4.1. Computational procedure 408
20.2.4.2. Choice of the regularization parameter 409
20.2.4.3. Alleviating spectral leakage due to the FFT 411
20.2.5. Representing and processing TFM data 411
20.2.5.1. Spatial maps of traction magnitude 411
20.2.5.2. Whole-cell traction 411
20.2.5.3. Traction along a predefined line 413
References 413
Chapter 21: Experimenters' guide to colocalization studies: finding a way through indicators and quantifiers, in practice 416
Introduction 417
21.1. An Overview of Colocalization Approaches 418
21.1.1. Two types of numerical values to extract: Colocalization Indicators and colocalization quantifiers 418
21.1.2. Two ways to work on colocalization evaluation: Taking the image as a whole and splitting it into objects 418
21.1.2.1. Working on image intensities 418
21.1.2.1.1. Legacy colocalization indicators and visualization methods 418
21.1.2.1.2. Legacy colocalization indicators and visualization methods, revisited 420
21.1.2.1.3. Which strategy to adopt? 422
21.1.2.2. Working on objects 424
21.1.2.2.1. Grouping pixels into objects: Image segmentation 424
21.1.2.2.2. Colocalization quantifiers based on object overlaps 425
21.1.2.2.3. Colocalization quantifiers based on object distances 426
21.1.2.2.4. Which strategy to adopt? 427
Conclusion 427
References 428
Chapter 22: User-friendly tools for quantifying the dynamics of cellular morphology and intracellular protein clusters 430
Introduction 431
22.1. Automated Classification of Cell Motion Types 432
22.2. GUI for Morphodynamics Classification and Ready Representation of Changes in Cell Behavior Over Time 436
22.3. Results of Morphodynamics Classification 438
22.4. Geometry-based Segmentation of Cells in Clusters 439
22.5. GUI for Cell Segmentation and Quantification of Protein Clusters 442
22.5.1. GUI module for 2D analysis 442
22.5.2. GUI module for 3D analysis 444
22.6. Results for Quantifying Protein Clusters 445
22.7. Discussion 445
Acknowledgments 447
References 447
Chapter 23: Ratiometric Imaging of pH Probes 450
Introduction 451
23.1. Currently Used Ratiometric pH Probes 451
23.1.1. pH-Sensitive ratiometric dyes 452
23.1.1.1. Advantages, limitations, and caveats of using dyes 454
23.1.2. Genetically encoded pH sensors 454
23.1.2.1. Advantages, limitations, and caveats of using genetically encoded pH biosensors 455
23.2. Applications 456
23.2.1. Measuring pHi in single cells 456
23.2.1.1. General considerations 456
23.2.1.2. Subcellular pH Measurements 457
23.2.2. Measuring pHi in tissues 459
23.3. Protocols 459
23.3.1. Solutions 459
HEPES buffer 459
HCO3 buffer 460
NH4Cl buffer 460
Nigericin buffer 461
23.3.2. Preparation of cultured cells 461
23.3.2.1. Dye loading in cultured cells 461
Materials required 461
23.3.2.2. Expression of genetically encoded pH biosensors in cultured cells 462
Materials required 462
23.3.2.3. Dye loading of whole-mount tissue 463
Materials required 463
Dissection tools 463
23.3.2.4. Expression of genetically coded pH biosensors in genetically tractable organisms 463
23.3.3. Ratiometric imaging 463
23.3.4. Generating nigericin calibration curves 464
Protocol 465
23.3.5. Ratiometric Analysis 465
Acknowledgments 466
References 466
Chapter 24: Toward quantitative fluorescence microscopy with DNA origami nanorulers 470
Introduction 471
24.1. The Principle of DNA Origami 473
24.2. Functionalizing DNA Origami Structures 473
24.3. DNA Origami as Fluorescence Microscopy Nanorulers 476
24.4. Brightness References Based on DNA Origami 478
24.5. Applications of DNA Origami Nanorulers for Visualizing Resolution 479
24.5.1. Nanorulers with defined distances for superresolution Microscopy 479
24.5.2. DNA-PAINT on the DNA origami nanoruler 480
24.6. How to Choose an Appropriate Nanoruler for a Given Application 482
References 484
Chapter 25: Imaging and physically probing kinetochores in live dividing cells 488
Introduction 489
The kinetochore 489
Mammalian cells: Challenges 490
Chapter overview 490
25.1. Spindle Compression to Image and Perturb Kinetochores 490
25.1.1. Historical context 490
25.1.2. Motivation 491
25.1.3. Methods 492
25.1.3.1. Choice of cell line 492
25.1.3.2. Cell culture 492
25.1.3.3. Agarose pad preparation 492
25.1.3.4. Experimental setup 493
25.1.3.5. Before spindle compression 493
25.1.3.6. Spindle compression 495
25.1.3.7. Choice of compression levels 495
25.1.3.8. After spindle compression 497
25.1.3.9. Troubleshooting tips 497
25.2. Imaging Kinetochore Dynamics at Subpixel Resolution Via Two-Color Reporter Probes 498
25.2.1. Historical context 498
25.2.2. Motivation 498
25.2.3. Methods 499
25.2.3.1. Gaussian fitting for subpixel resolution 499
25.2.3.2. Choice of cell line and reporter probes 499
25.2.3.3. Expression of reporter probes 500
25.2.3.4. Experimental setup 502
25.2.3.5. Before live cell imaging: Two-color bead registration 502
25.2.3.6. Subpixel resolution kinetochore imaging via two-color reporter probes 503
25.2.3.7. Data analysis for subpixel resolution kinetochore imaging 503
25.2.3.8. Key considerations for interpretation of interprobe distances 504
Conclusion and Outlook 505
Acknowledgments 506
References 506
Chapter 26: Adaptive fluorescence microscopy by online feedback image analysis 510
Introduction 511
26.1. Requirements for Adaptive Feedback Microscopy 513
26.2. Selected Applications 514
26.2.1. Automated detection and imaging of Plasmodium-infected cells 514
26.2.1.1. Motivation 514
26.2.1.2. Sample preparation 516
26.2.1.3. Feedback microscopy implementation using CellProfiler and LASAF Matrix Screener 516
26.2.1.4. CellProfiler for adaptive feedback microscopy 516
26.2.1.5. Setting up and running the experiment 517
26.2.1.6. Results and discussion 518
26.2.2. Automated FRAP on ER exit sites 518
26.2.2.1. Motivation and automation workflow overview 518
26.2.2.2. Sample preparation 519
26.2.2.3. Workflow and implementation 519
26.2.2.4. Results and discussion 521
Acknowledgments 522
References 522
Chapter 27: Open-source solutions for SPIMage processing 526
Introduction 527
Brief overview of light sheet microscopy flavors 527
Applications of light sheet microscopy 529
27.1. Prerequisites 530
27.1.1. Parameters of example dataset 530
27.1.2. Fluorescent beads as fiducial markers for registration 531
27.1.3. Installation and configuration of Fiji 531
27.1.4. Hardware requirements 532
27.1.5. File formats preprocessing and naming conventions 532
27.2. Overview of the SPIM Image-Processing Pipeline 533
27.3. Bead-based Registration 534
27.3.1. Workflow 536
27.3.2. Results 538
27.4. Multiview Fusion 539
27.4.1. Content-Based Multiview Fusion 539
27.4.1.1. Workflow 539
27.4.1.2. Results 542
27.4.2. Multiview Deconvolution 542
27.4.2.1. Workflow 543
27.4.2.2. Results 544
27.5. Processing on a High-Performance Cluster 545
27.6. Future Applications 546
References 548
Chapter 28: Second-harmonic generation imaging of cancer 552
Introduction 553
28.1. SHG Physical and Chemical Background 553
28.2. SHG Instrumentation 554
28.3. Collagen Structure as a Biomarker 554
28.4. SHG in Cancer Research 556
28.4.1. Breast cancer 556
28.4.2. Ovarian cancer 558
28.4.3. Skin cancer 558
28.4.4. SHG research in other types of cancer 560
28.5. Quantitative Analysis of SHG Images 562
Conclusion 562
References 563
Index 568
Volumes in Series 578
Color Plate 590