Current Protocols Select

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