Table of Contents
Foreword xiii
Preface xv
Contributors xvii
Chapter 1 Fundamentals of the Microscope
Introduction 3
Fluorescence Microscopy: A Concise Guide to Current Imaging Methods 5
Introduction 5
Wide-Field Fluorescence Microscopy (WFFM) Techniques 6
Modern Confocal Microscopy 9
Total Internal Reflection Fluorescence (TIRF) Microscopy 12
Two-Photon Fluorescence Microscopy (TPFM) 14
Stimulated Emission Depletion (STED) Fluorescence Microscopy 16
Final Considerations 18
Acknowledgements 19
Literature Cited 19
Microscope Objectives 21
Introduction 21
Image Fidelity 21
Properties of Microscope Objectives 25
Construction and Types of Microscope Objectives 26
Modern Objectives 28
Objectives for Other Microscopy Applications 32
Other Considerations in Choosing Objectives 33
Literature Cited 34
Key References 34
Internet Resources 34
Light Microscopy Digital Imaging 35
History of Microscopy Image Capture 35
Solid-State Sensors 35
Spectral Sensitivity of Sensors 37
Camera Noise 38
Coupling Digital Cameras to Microscopes 40
Color Imaging 42
Camera and Sensor Characteristics 43
Modes of Image Capture 44
Microscope Optimization for Digital Imaging 45
Care and Maintenance 45
Key References 47
Optical Filters for Wavelength Selection in Fluorescence Instrumentation 49
Introduction 49
Optical Thin-Film Interference Filters 49
Optical Filter Configurations in Fluorescence Instruments 52
Fluorescence Filters Impact Optical System Performance 63
Tunable Optical Filters 71
Conclusion 75
Literature Cited 76
Proper Alignment and Adjustment of the Light Microscope 77
Major Components of the Light Microscope 78
Basic Imaging and K¨ohler Illumination Light Paths for Bright-Field, Fluorescence, and Dark-Field Microscopy 83
Basic Imaging for Dark-Field Microscopy 85
Basic Protocol 1: Alignment for K¨ohler Illumination in Bright-Field, Transmitted Light Microscopy 86
Basic Protocol 2: Alignment of the Eyepieces 89
Basic Protocol 3: Alignment for K¨ohler Illumination in Epifluorescence Microscopy 90
Basic Protocol 4: Alignment for Phase-Contrast Microscopy 92
Basic Protocol 5: Alignment for DIC Microscopy 94
Alignment for Dark-Field Microscopy 98
Basic Protocol 6: Alignment for Low-Power Magnification Dark-Field Microscopy 99
Basic Protocol 7: Alignment for High-Power Magnification Dark-Field Illumination 100
Support Protocol 1: Matching Microscope Magnification to Detector Resolution 101
Support Protocol 2: Calibrating Image Magnification with a Stage Micrometer 102
Tests for the Optical Performance of the Microscope 103
Support Protocol 3: Testing Phase-Contrast and DIC Using Diatom Testing Slide 103
Support Protocol 4: Testing Phase-Contrast, Dark-Field, and DIC Microscopes Using a Squamous Cheek Cell Test Slide 103
Support Protocol 5: Testing Fluorescence Using a Red, Green, and Blue Fluorescent Tissue Culture Cell Test Slide 103
Support Protocol 6: Care and Cleaning of Microscope Optics 105
Commentary 106
Literature Cited 107
Chapter 2 Basic Methods
Introduction 111
Section I Sample Preparation for Conventional Microscopy
Cryosectioning 113
Basic Protocol: Specimen Preparation and Sectioning 113
Support Protocol 1: Tissue Fixation and Sucrose Infusion 117
Support Protocol 2: Perfusion of Adult Mice 117
Reagents and Solutions 118
Commentary 119
Literature Cited 120
Immunohistochemistry 121
Introduction 121
Basic Protocol 1: Immunofluorescent Labeling of Cells Grown as Monolayers 121
Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells 123
Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections 124
Alternate Protocol 2: Immunofluorescent Labeling Using Streptavidin-Biotin Conjugates 125
Alternate Protocol 3: Immunofluorescent Double-Labeling of Tissue Sections 126
Reagents and Solutions 127
Commentary 127
Literature Cited 131
Section II Dyes and Probes
A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures 133
Introduction 133
Basic Protocol 1: BacMam Constructs 136
Alternate Protocol 1: Non-Pseudo-Typed BacMam Viruses/Hard-To-Transduce Cell Types 140
Basic Protocol 2: Actin Labeling 141
Basic Protocol 3: Autophagosome Labeling by Transduction of Cells with Premo Autophagy Sensor GFP-LC3B 142
Alternate Protocol 2: Performing Autophagosome Labeling with an Antibody 143
Basic Protocol 4: Wheat Germ Agglutinin Conjugates for Plasma Membrane Labeling 145
Basic Protocol 5: Endoplasmic Reticulum and Nuclear Membrane Labeling Using ER-Tracker Reagents 145
Basic Protocol 6: Labeling Endosomes with pHrodo 10k Dextran 146
Basic Protocol 7: Labeling Golgi Apparatus Using Dye-Labeled Ceramides 147
Basic Protocol 8: Labeling Lysosomes Using LysoTracker Red DND-99 149
Basic Protocol 9: Labeling Mitochondria Using MitoTracker Red CMXRos 150
Basic Protocol 10: Labeling Nucleoli Using SYTO RNASelect Green 152
Basic Protocol 11: Labeling Peroxisomes Using CellLight BacMam 2.0 Peroxisomes-GFP 153
Alternate Protocol 3: Labeling Peroxisomes Using Antibodies 154
Basic Protocol 12: Labeling Tubulin Microtubules with TubulinTracker Green 156
Basic Protocol 13: Labeling Whole Cells or Cytoplasm with 5(6)-CFDA SE 156
Reagents and Solutions 158
Commentary 161
Literature Cited 197
Internet Resources 203
The Fluorescent Protein Color Palette 207
Introduction 207
Fluorescent Protein Brightness and Maturation 210
Phototoxicity and Photostability 212
Oligomerization 214
The Fluorescent Protein Color Palette 216
Optical Highlighter Fluorescent Proteins 232
The Future of Fluorescent Proteins 239
Literature Cited 239
Photoactivation and Imaging of Optical Highlighter Fluorescent Proteins 247
Introduction 247
Background 247
Requirements for Highlighting Fluorescent Proteins 252
Optimization Procedures 253
General Photoactivation Experiment 255
Uses of Optical Highlighter Fluorescent Proteins 256
Application of Optical Highlighter Fluorescent Proteins in Cytometry 258
Future Directions of Optical Highlighter Fluorescent Proteins 258
Acknowledgement 259
Literature Cited 259
Section III Optical Sectioning Microscopy
Basic Confocal Microscopy 261
Introduction 261
Basis of Optical Sectioning 263
Configuration of an LSCM 265
Practical Guidelines 268
Commentary 275
Acknowledgements 278
Literature Cited 278
Key References 280
Internet Resources 280
Evaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous Factors to Consider 283
Introduction 283
Applications 285
Hardware 286
Software 288
Quality Assurance (QA) 289
Service, Support, and Company 293
Maintenance/Cleanup Protocol 294
Price 294
Recommendation from Colleagues 294
Summary and Conclusions 294
Disclaimer 295
Resources Listed 295
Acknowledgements 295
Literature Cited 295
3D Deconvolution Microscopy 297
Introduction 297
Image Formation 297
Resolution and Sampling 301
Estimating and Optimizing the PSF 302
Deblurring and Deconvolution Algorithms 303
Blind Deconvolution 306
Example Deconvolution Results 307
Deconvolution Software 309
Basic Protocol: Data Acquisition and Deconvolution Analysis 312
Concluding Remarks 315
Literature Cited 315
Key References 316
Internet Resources 316
Multi-Photon Imaging 317
Introduction 317
Multi-Photon Microscopy 317
Multi-Photon Imaging in Practice 323
Concluding Remarks 328
Literature Cited 328
Chapter 3 Applications
Introduction 333
Section I Basic Live Cell Imaging
Building a Live-Cell Microscope: What You Need and How to Do It 335
Defining the System 335
Building a Live-Cell Scope: Components and Considerations 337
Transmitted Light Choices 344
Summary 346
Time-Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single-Cell Level 347
Introduction 347
System Setup 348
Basic Protocol 1: Time-Lapse Acquisition Using Adherent Cells 349
Alternate Protocol 1: Time-Lapse Acquisition with Endpoint Assay to Mark S-Phase Cells 350
Alternate Protocol 2: Time-Lapse Acquisition Using Suspension Cells 351
Basic Protocol 2: Sequence Analysis for Mitosis Event or Cell Death 352
Basic Protocol 3: Data Mining - Normalized Event Distribution 353
Basic Protocol 4: Data Mining - Time-to-Event Curves 354
Basic Protocol 5: Data Mining - Duration of Mitotic Event 355
Basic Protocol 6: Data Mining - G2 Checkpoint Breaching 355
Basic Protocol 7: Data Mining - Deriving Basic Lineage Parameters 356
Commentary 356
Literature Cited 359
Internet Resources 360
Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches 361
Introduction 361
Strategic Planning 361
Basic Protocol 1: High-Resolution z-Stack and Time-Lapse Imaging of Mitochondria 363
Alternate Protocol: Imaging Mitochondrial Morphology Alterations 366
Basic Protocol 2: Fluorescence Recovery After Photobleaching on Mitochondria 367
Basic Protocol 3: Microirradiation Assay to Assess Electrical Continuity in Mitochondria 372
Support Protocol: Staining Mitochondria in Live Cells to Assess Mitochondrial Function by Imaging 375
Commentary 378
Literature Cited 382
Analysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching (FRAP) 385
Introduction 385
Basic Protocol 1: How to Set Up a FRAP Experiment 387
Basic Protocol 2: Confocal FRAP Measurements of the Lateral Diffusion of Plasma Membrane Proteins and Lipids 391
Alternate Protocol 1: Lateral Diffusion Measurements for a Rapidly Diffusing Soluble Protein 393
Alternate Protocol 2: FRAP Analysis of Intracellular Trafficking Kinetics 395
Basic Protocol 3: Working with FRAP Data 397
Basic Protocol 4: Further Analysis of FRAP Data to Obtain Diffusion Coefficients 399
Commentary 401
Acknowledgements 411
Literature Cited 411
Confocal Imaging of Cell Division 415
Introduction 415
Spinning Disk Confocal 415
Confocal Imaging of Chromosome Condensation in C. elegans Embryos 420
Confocal Imaging of Spindle Assembly and Chromosome Dynamics 421
Confocal Imaging of Cytokinesis 424
Discussion 425
Acknowledgements 426
Literature Cited 426
Total Internal Reflection Fluorescence (TIRF) Microscopy 429
Introduction 429
The Theory Behind the Technique 430
TIRF Objectives 432
Empirically Determining Incident Angle/Penetration Depth 434
TIRF Imaging of Plasma Membrane Receptors in Neurons 436
Multi-Wavelength TIRFM 438
Final Experimental Suggestions 441
Concluding Remarks 442
Literature Cited 442
Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events 445
Introduction 445
Basic Protocol 1: Through-the-Objective TIRF Protocol 445
Alternate Protocol: Improved Uniformity in the Excitation Field Protocol 450
Basic Protocol 2: Through-the-Prism TIRF Protocol 452
Commentary 454
Literature Cited 465
Section II Fluorescence Resonance Energy Transfer
Imaging Protein-Protein Interactions by F¨orster Resonance Energy Transfer (FRET) Microscopy in Live Cells 467
Commentary 474
Literature Cited 479
Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy 481
Basic Protocol: FRET Microscopy of Fixed Cells 482
Support Protocol 1: Nuclear and Cytosolic Microinjection 485
Support Protocol 2: Protein Labeling with Cy3 487
Reagents and Solutions 490
Commentary 490
Literature Cited 496
Use of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide–Based Signaling Events in Isolated Perfused Lung 499
Introduction 499
Strategic Planning 499
Basic Protocol 1: Isolating and Perfusing Mouse Lung 500
Basic Protocol 2: No-Induced Protein Modifications Detected by FRET Using Spectral Confocal Microscopy 503
Reagents and Solutions 506
Commentary 507
Literature Cited 510
Section III Imaging of Model Systems
Fluorescence Imaging Techniques for Studying Drosophila Embryo Development 513
Introduction 513
Strategic Planning 514
Basic Protocol 1: Generation of Transgenic Drosophila for Live Fluorescence Microscopy Using the Gal4/UAS System 525
Basic Protocol 2: Preparation of Drosophila Embryos for Fluorescence Microscopy 529
Basic Protocol 3: Time-Lapse Confocal Imaging of Living Drosophila Embryos 531
Basic Protocol 4: Time-Lapse Imaging of Living Drosophila Embryos with Two-Photon Laser Scanning Microscopy 537
Basic Protocol 5: Fluorescence Recovery After Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope
Capable of Selective Photobleaching 540
Basic Protocol 6: Fluorescence Loss in Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching 546
Basic Protocol 7: Photoactivation in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching 548
Reagents and Solutions 553
Commentary 553
Literature Cited 557
Time-Lapse Imaging of Embryonic Neural Stem Cell Division in Drosophila by Two-Photon Microscopy 561
Introduction 561
Basic Protocol: Time-Lapse Imaging by Two-Photon Microscopy 561
Support Protocol: Embryo Preparation 564
Commentary 565
Acknowledgements 569
Literature Cited 569
Imaging Tumor Cell Movement In Vivo 571
Introduction 571
Basic Protocol 1: Generation and In Vivo Imaging of Mammary Tumors 571
Support Protocol 1: In Vivo Imaging Microscope Setup 579
Support Protocol 2: Labeling Vasculature and Macrophages 580
Support Protocol 3: Blood Vessel Imaging Using an Indwelling Catheter 581
Support Protocol 4: Second Harmonic Fiber Imaging 583
Basic Protocol 2: Multiphoton Time-Lapse Image Analysis Using ImageJ and Custom Plugins 583
Support Protocol 5: Separation of Spectral Overlap 586
Reagents and Solutions 587
Commentary 587
Literature Cited 589
Live-Animal Imaging of Renal Function by Multiphoton Microscopy 591
Introduction 591
Basic Protocol 1: Glomerular Permeability 592
Basic Protocol 2: Proximal Tubule Endocytosis 593
Basic Protocol 3: Vascular Flow 594
Basic Protocol 4: Vascular Permeability 596
Basic Protocol 5: Mitochondrial Function 597
Basic Protocol 6: Apoptosis 598
Support Protocol: Anesthesia and Surgical Creation of a Retroperitoneal Surgical Window for Intravital Imaging 599
Reagents and Solutions 602
Commentary 602
Literature Cited 608
Biological Second and Third Harmonic Generation Microscopy 611
Strategic Planning 612
Basic Protocol 1: Designing a Microscope System for HHGM 612
Basic Protocol 2: Detection of Fibrillar Collagen in Connective Tissue Ex Vivo 619
Basic Protocol 3: Detection of SHG in Mouse Tissues by Intravital Microscopy 621
Basic Protocol 4: Simultaneous Detection of Cells and Collagen Fibers In Vitro and In Vivo 622
Support Protocol 1: Cytoplasmic Staining of Live Cells 625
Support Protocol 2: Establishment of 3-D Collagen Cultures 625
Reagents and Solutions 626
Commentary 626
Acknowledgements 632
Literature Cited 632
Two-Photon Imaging of the Immune System 635
Introduction 635
Basic Protocol 1: Preparing the Thymus of a Mouse for Two-Photon Imaging 636
Basic Protocol 2: Preparing the Mesenteric Lymph Nodes (MLNs) of a Mouse for Two-Photon Imaging 637
Basic Protocol 3: Preparing Segments from the Intestine of a Mouse for Two-Photon Imaging 639
Alternate Protocol 1: Agarose Embedding of a Small Tissue Sample or Organotypic Cultures 640
Alternate Protocol 2: Preparing Thymic Slices for Two-Photon Imaging 642
Alternate Protocol 3: Overlaying Thymic Slices with Fluorescently Labeled Cells 645
Support Protocol: Setting Up Two-Photon Imaging Conditions 646
Reagents and Solutions 647
Commentary 647
Literature Cited 654
Section IV Super-Resolution Methods
Super-Resolution Microscopy: A Comparative Treatment 657
Introduction 657
Super-Resolution Imaging Methodologies 657
Point-Spread Function Engineering 668
Concluding Remarks 677
Acknowledgements 677
Literature Cited 677
Photoactivated Localization Microscopy (PALM) of Adhesion Complexes 683
Introduction 683
Strategic Planning 683
Basic Protocol 1: Preparing PALM Instrumentation 687
Basic Protocol 2: PALM Imaging tdEos/Paxillin Distributions in Fixed Cells 697
Basic Protocol 3: Dual-Color PALM Imaging of tdEos/Vinculin and Dronpa α-Actinin in Fixed Cells 701
Support Protocol 1: Preparing Clean Coverslips 704
Support Protocol 2: Transfection of tdEos/Paxillin into HFF-1 Cells 705
Reagents and Solutions 707
Commentary 708
Literature Cited 710
Comparative and Practical Aspects of Localization-Based Super-Resolution Imaging 713
Introduction 713
Basic Protocol 1: Multi-Channel Labeling of Microtubules and Mitochondria with STORM Tandem Dye Pairs 713
Support Protocol 1: Dye Preparation and Secondary Antibody Labeling 715
Basic Protocol 2: Buffer and Imaging Conditions for Synthetic Photoswitchable Dyes 716
Basic Protocol 3: Labeling Proteins via SNAP Tags for Live-Cell Localization Super Resolution 717
Support Protocol 2: Buffer and Imaging Conditions for Live-Cell Localization Super Resolution 719
Commentary 719
Acknowledgements 723
Literature Cited 723
Chapter 4 Image Processing
Introduction 727
Ethical Considerations When Altering Digital Images 729
Introduction 729
Golden Rules 729
Guidelines from Specific Journals 731
Literature Cited 733
From Image to Data Using Common Image-Processing Techniques 735
Introduction 735
Image Anatomy 735
Image Processing 736
Concluding Remarks 751
Literature Cited 751
Practical Considerations When Altering Digital Images 753
Introduction 753
Sampling Resolution 753
Resampling 755
Acquiring Images 758
Photoshop and Scientific Image–Analysis Programs 762
Optimizing the Display 764
Using Images from Vector Programs and PowerPoint 765
Altering Images Using Photoshop 766
Inserting Files into PowerPoint 784
Literature Cited 785
Appendix 1: Common Stock Solutions, Buffers, and Media 787
Index 791
