Librería Servicio Médico

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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