Current Protocols Select: Methods and Applications in Microscopy and Imaging / Edition 1

Current Protocols Select: Methods and Applications in Microscopy and Imaging / Edition 1

by Simon Watkins, Claudette St. Croix

The advanced light microscope is a highly sophisticated robotic tool which, when coupled with appropriate detectors and image processing, allows scientists to investigate the inner workings of biological systems from the single molecule, through cells, to the entire living organism. The last twenty-five years have brought significant growth in the utility of

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The advanced light microscope is a highly sophisticated robotic tool which, when coupled with appropriate detectors and image processing, allows scientists to investigate the inner workings of biological systems from the single molecule, through cells, to the entire living organism. The last twenty-five years have brought significant growth in the utility of the microscope, especially given novel imaging tools (e.g., the computerized microscope and the confocal microscope), reagents (fluorescent proteins), and powerful computers that can process enormous quantities of data produced by these technologies. Today, the application of the light microscope is no longer a limited descriptive field but a battery of extremely complex, rich, and useful primary research tools. It is evident that the successful integration of these technologies is fundamental to bench biomedical research.

This book is intended to be a resource for those new to the use of the microscope as well as for seasoned investigators, exposing them to the wealth of opportunities to visualize the molecules and cellular processes they are studying. Because these are Current Protocols articles, they provide detailed step-by-step instructions for the various methods and applications, described and written at a level that lets every investigator employ even very sophisticated microscopy methods.

  • For graduate students, postdocs, and individuals who are branching into new areas as an entry into the specialized techniques of microscopy
  • A resource that encourages the use of the microscope beyond that of the simple light microscope
  • An essential reference for core facilities at academic institutions
  • An "instructional" text essential for each of these core resources

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Table of Contents

Foreword xiii

Preface xv

Contributors xvii

Chapter 1 Fundamentals of the Microscope

Introduction 3

Fluorescence Microscopy: A Concise Guide to Current ImagingMethods 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 Microscopy16

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 FluorescenceInstrumentation 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 Kóhler Illumination Light Paths forBright-Field, Fluorescence, and Dark-Field Microscopy 83

Basic Imaging for Dark-Field Microscopy 85

Basic Protocol 1: Alignment for Kóhler Illumination inBright-Field, Transmitted Light Microscopy 86

Basic Protocol 2: Alignment of the Eyepieces 89

Basic Protocol 3: Alignment for Kóhler Illumination inEpifluorescence 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 MagnificationDark-Field Microscopy 99

Basic Protocol 7: Alignment for High-Power MagnificationDark-Field Illumination 100

Support Protocol 1: Matching Microscope Magnification toDetector Resolution 101

Support Protocol 2: Calibrating Image Magnification with a StageMicrometer 102

Tests for the Optical Performance of the Microscope 103

Support Protocol 3: Testing Phase-Contrast and DIC Using DiatomTesting Slide 103

Support Protocol 4: Testing Phase-Contrast, Dark-Field, and DICMicroscopes Using a Squamous Cheek Cell Test Slide 103

Support Protocol 5: Testing Fluorescence Using a Red, Green, andBlue Fluorescent Tissue Culture Cell Test Slide 103

Support Protocol 6: Care and Cleaning of Microscope Optics105

Commentary 106

Literature Cited 107

Chapter 2 Basic Methods

Introduction 111

Section I Sample Preparation for ConventionalMicroscopy

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 asMonolayers 121

Alternate Protocol 1: Immunofluorescent Labeling of SuspensionCells 123

Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections124

Alternate Protocol 2: Immunofluorescent Labeling UsingStreptavidin-Biotin Conjugates 125

Alternate Protocol 3: Immunofluorescent Double-Labeling ofTissue Sections 126

Reagents and Solutions 127

Commentary 127

Literature Cited 131

Section II Dyes and Probes

A Review of Reagents for Fluorescence Microscopy of CellularCompartments and Structures 133

Introduction 133

Basic Protocol 1: BacMam Constructs 136

Alternate Protocol 1: Non-Pseudo-Typed BacMamViruses/Hard-To-Transduce Cell Types 140

Basic Protocol 2: Actin Labeling 141

Basic Protocol 3: Autophagosome Labeling by Transduction ofCells with Premo Autophagy Sensor GFP-LC3B 142

Alternate Protocol 2: Performing Autophagosome Labeling with anAntibody 143

Basic Protocol 4: Wheat Germ Agglutinin Conjugates for PlasmaMembrane Labeling 145

Basic Protocol 5: Endoplasmic Reticulum and Nuclear MembraneLabeling Using ER-Tracker Reagents 145

Basic Protocol 6: Labeling Endosomes with pHrodo 10k Dextran146

Basic Protocol 7: Labeling Golgi Apparatus Using Dye-LabeledCeramides 147

Basic Protocol 8: Labeling Lysosomes Using LysoTracker RedDND-99 149

Basic Protocol 9: Labeling Mitochondria Using MitoTracker RedCMXRos 150

Basic Protocol 10: Labeling Nucleoli Using SYTO RNASelect Green152

Basic Protocol 11: Labeling Peroxisomes Using CellLight BacMam2.0 Peroxisomes-GFP 153

Alternate Protocol 3: Labeling Peroxisomes Using Antibodies154

Basic Protocol 12: Labeling Tubulin Microtubules withTubulinTracker Green 156

Basic Protocol 13: Labeling Whole Cells or Cytoplasm with5(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 FluorescentProteins 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 inCytometry 258

Future Directions of Optical Highlighter Fluorescent Proteins258

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 ofthe 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 Analysis312

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 It335

Defining the System 335

Building a Live-Cell Scope: Components and Considerations337

Transmitted Light Choices 344

Summary 346

Time-Lapse Microscopy Approaches to Track Cell Cycle and LineageProgression at the Single-Cell Level 347

Introduction 347

System Setup 348

Basic Protocol 1: Time-Lapse Acquisition Using Adherent Cells349

Alternate Protocol 1: Time-Lapse Acquisition with Endpoint Assayto Mark S-Phase Cells 350

Alternate Protocol 2: Time-Lapse Acquisition Using SuspensionCells 351

Basic Protocol 2: Sequence Analysis for Mitosis Event or CellDeath 352

Basic Protocol 3: Data Mining—Normalized EventDistribution 353

Basic Protocol 4: Data Mining—Time-to-Event Curves 354

Basic Protocol 5: Data Mining—Duration of Mitotic Event355

Basic Protocol 6: Data Mining—G2 Checkpoint Breaching355

Basic Protocol 7: Data Mining—Deriving Basic LineageParameters 356

Commentary 356

Literature Cited 359

Internet Resources 360

Analysis of Mitochondrial Dynamics and Functions Using ImagingApproaches 361

Introduction 361

Strategic Planning 361

Basic Protocol 1: High-Resolution z-Stack and Time-Lapse Imagingof Mitochondria 363

Alternate Protocol: Imaging Mitochondrial Morphology Alterations366

Basic Protocol 2: Fluorescence Recovery After Photobleaching onMitochondria 367

Basic Protocol 3: Microirradiation Assay to Assess ElectricalContinuity in Mitochondria 372

Support Protocol: Staining Mitochondria in Live Cells to AssessMitochondrial Function by Imaging 375

Commentary 378

Literature Cited 382

Analysis of Protein and Lipid Dynamics Using ConfocalFluorescence 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 LateralDiffusion of Plasma Membrane Proteins and Lipids 391

Alternate Protocol 1: Lateral Diffusion Measurements for aRapidly Diffusing Soluble Protein 393

Alternate Protocol 2: FRAP Analysis of Intracellular TraffickingKinetics 395

Basic Protocol 3: Working with FRAP Data 397

Basic Protocol 4: Further Analysis of FRAP Data to ObtainDiffusion 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. elegansEmbryos 420

Confocal Imaging of Spindle Assembly and Chromosome Dynamics421

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) MicroscopyIlluminator 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 FieldProtocol 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 Fórster ResonanceEnergy Transfer (FRET) Microscopy in Live Cells 467

Commentary 474

Literature Cited 479

Imaging Protein-Protein Interactions by Fluorescence ResonanceEnergy 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 DetectNitric Oxide–Based Signaling Events in Isolated Perfused Lung499

Introduction 499

Strategic Planning 499

Basic Protocol 1: Isolating and Perfusing Mouse Lung 500

Basic Protocol 2: No-Induced Protein Modifications Detected byFRET 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 EmbryoDevelopment 513

Introduction 513

Strategic Planning 514

Basic Protocol 1: Generation of Transgenic Drosophila for LiveFluorescence Microscopy Using the Gal4/UAS System 525

Basic Protocol 2: Preparation of Drosophila Embryos forFluorescence Microscopy 529

Basic Protocol 3: Time-Lapse Confocal Imaging of LivingDrosophila Embryos 531

Basic Protocol 4: Time-Lapse Imaging of Living DrosophilaEmbryos with Two-Photon Laser Scanning Microscopy 537

Basic Protocol 5: Fluorescence Recovery After Photobleaching inLiving Drosophila Embryos Using a Laser Scanning ConfocalMicroscope

Capable of Selective Photobleaching 540

Basic Protocol 6: Fluorescence Loss in Photobleaching in LivingDrosophila Embryos Using a Laser Scanning Confocal MicroscopeCapable of Selective Photobleaching 546

Basic Protocol 7: Photoactivation in Living Drosophila EmbryosUsing a Laser Scanning Confocal Microscope Capable of SelectivePhotobleaching 548

Reagents and Solutions 553

Commentary 553

Literature Cited 557

Time-Lapse Imaging of Embryonic Neural Stem Cell Division inDrosophila by Two-Photon Microscopy 561

Introduction 561

Basic Protocol: Time-Lapse Imaging by Two-Photon Microscopy561

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 MammaryTumors 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 IndwellingCatheter 581

Support Protocol 4: Second Harmonic Fiber Imaging 583

Basic Protocol 2: Multiphoton Time-Lapse Image Analysis UsingImageJ 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 Microscopy591

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 aRetroperitoneal Surgical Window for Intravital Imaging 599

Reagents and Solutions 602

Commentary 602

Literature Cited 608

Biological Second and Third Harmonic Generation Microscopy611

Strategic Planning 612

Basic Protocol 1: Designing a Microscope System for HHGM 612

Basic Protocol 2: Detection of Fibrillar Collagen in ConnectiveTissue Ex Vivo 619

Basic Protocol 3: Detection of SHG in Mouse Tissues byIntravital Microscopy 621

Basic Protocol 4: Simultaneous Detection of Cells and CollagenFibers In Vitro and In Vivo 622

Support Protocol 1: Cytoplasmic Staining of Live Cells 625

Support Protocol 2: Establishment of 3-D Collagen Cultures625

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-PhotonImaging 636

Basic Protocol 2: Preparing the Mesenteric Lymph Nodes (MLNs) ofa Mouse for Two-Photon Imaging 637

Basic Protocol 3: Preparing Segments from the Intestine of aMouse for Two-Photon Imaging 639

Alternate Protocol 1: Agarose Embedding of a Small Tissue Sampleor Organotypic Cultures 640

Alternate Protocol 2: Preparing Thymic Slices for Two-PhotonImaging 642

Alternate Protocol 3: Overlaying Thymic Slices withFluorescently Labeled Cells 645

Support Protocol: Setting Up Two-Photon Imaging Conditions646

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 AdhesionComplexes 683

Introduction 683

Strategic Planning 683

Basic Protocol 1: Preparing PALM Instrumentation 687

Basic Protocol 2: PALM Imaging tdEos/Paxillin Distributions inFixed Cells 697

Basic Protocol 3: Dual-Color PALM Imaging of tdEos/Vinculin andDronpa α-Actinin in Fixed Cells 701

Support Protocol 1: Preparing Clean Coverslips 704

Support Protocol 2: Transfection of tdEos/Paxillin into HFF-1Cells 705

Reagents and Solutions 707

Commentary 708

Literature Cited 710

Comparative and Practical Aspects of Localization-BasedSuper-Resolution Imaging 713

Introduction 713

Basic Protocol 1: Multi-Channel Labeling of Microtubules andMitochondria with STORM Tandem Dye Pairs 713

Support Protocol 1: Dye Preparation and Secondary AntibodyLabeling 715

Basic Protocol 2: Buffer and Imaging Conditions for SyntheticPhotoswitchable Dyes 716

Basic Protocol 3: Labeling Proteins via SNAP Tags for Live-CellLocalization Super Resolution 717

Support Protocol 2: Buffer and Imaging Conditions for Live-CellLocalization 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 Techniques735

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

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