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Current Protocols Select: Methods and Applications in Microscopy and Imaging - Simon Watkins / Claudette St. Croix

Current Protocols Select: Methods and Applications in Microscopy and Imaging - Simon Watkins / Claudette St. Croix

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Author : Simon Watkins / Claudette St. Croix Language: English Finishing : Papaerback, 824 pages ISBN : 978-1-118-04431-5 Edition Number: 2014 Description: Compiled by editors with hands-on experience in microscopy, teaching, and protocol design and communication, this book provides a practical, bench-side guide to the various methods and applications of the advanced light microscope in the...
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Descripción completa de: Current Protocols Select: Methods and Applications in Microscopy and Imaging - Simon Watkins / Claudette St. Croix

Author : Simon Watkins / Claudette St. Croix

Language: English

Finishing : Papaerback, 824 pages

ISBN : 978-1-118-04431-5

Edition Number: 2014

Description:

Compiled by editors with hands-on experience in microscopy, teaching, and protocol design and communication, this book provides a practical, bench-side guide to the various methods and applications of the advanced light microscope in the cell biology laboratory. It offers detailed step-by-step instructions written at a level that lets investigators employ even very sophisticated microscopy methods. The result is a resource for seasoned investigators and those new to the use of the microscope alike.

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

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