{"product_id":"current-protocols-select-isbn-9781118044315","title":"Current Protocols Select","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.  \u003cp\u003eForeword xiii\u003c\/p\u003e \u003cp\u003ePreface xv\u003c\/p\u003e \u003cp\u003eContributors xvii\u003c\/p\u003e \u003cp\u003e\u003cb\u003eChapter 1 Fundamentals of the Microscope\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eIntroduction 3\u003c\/p\u003e \u003cp\u003eFluorescence Microscopy: A Concise Guide to Current Imaging Methods 5\u003c\/p\u003e \u003cp\u003eIntroduction 5\u003c\/p\u003e \u003cp\u003eWide-Field Fluorescence Microscopy (WFFM) Techniques 6\u003c\/p\u003e \u003cp\u003eModern Confocal Microscopy 9\u003c\/p\u003e \u003cp\u003eTotal Internal Reflection Fluorescence (TIRF) Microscopy 12\u003c\/p\u003e \u003cp\u003eTwo-Photon Fluorescence Microscopy (TPFM) 14\u003c\/p\u003e \u003cp\u003eStimulated Emission Depletion (STED) Fluorescence Microscopy 16\u003c\/p\u003e \u003cp\u003eFinal Considerations 18\u003c\/p\u003e \u003cp\u003eAcknowledgements 19\u003c\/p\u003e \u003cp\u003eLiterature Cited 19\u003c\/p\u003e \u003cp\u003eMicroscope Objectives 21\u003c\/p\u003e \u003cp\u003eIntroduction 21\u003c\/p\u003e \u003cp\u003eImage Fidelity 21\u003c\/p\u003e \u003cp\u003eProperties of Microscope Objectives 25\u003c\/p\u003e \u003cp\u003eConstruction and Types of Microscope Objectives 26\u003c\/p\u003e \u003cp\u003eModern Objectives 28\u003c\/p\u003e \u003cp\u003eObjectives for Other Microscopy Applications 32\u003c\/p\u003e \u003cp\u003eOther Considerations in Choosing Objectives 33\u003c\/p\u003e \u003cp\u003eLiterature Cited 34\u003c\/p\u003e \u003cp\u003eKey References 34\u003c\/p\u003e \u003cp\u003eInternet Resources 34\u003c\/p\u003e \u003cp\u003eLight Microscopy Digital Imaging 35\u003c\/p\u003e \u003cp\u003eHistory of Microscopy Image Capture 35\u003c\/p\u003e \u003cp\u003eSolid-State Sensors 35\u003c\/p\u003e \u003cp\u003eSpectral Sensitivity of Sensors 37\u003c\/p\u003e \u003cp\u003eCamera Noise 38\u003c\/p\u003e \u003cp\u003eCoupling Digital Cameras to Microscopes 40\u003c\/p\u003e \u003cp\u003eColor Imaging 42\u003c\/p\u003e \u003cp\u003eCamera and Sensor Characteristics 43\u003c\/p\u003e \u003cp\u003eModes of Image Capture 44\u003c\/p\u003e \u003cp\u003eMicroscope Optimization for Digital Imaging 45\u003c\/p\u003e \u003cp\u003eCare and Maintenance 45\u003c\/p\u003e \u003cp\u003eKey References 47\u003c\/p\u003e \u003cp\u003eOptical Filters for Wavelength Selection in Fluorescence Instrumentation 49\u003c\/p\u003e \u003cp\u003eIntroduction 49\u003c\/p\u003e \u003cp\u003eOptical Thin-Film Interference Filters 49\u003c\/p\u003e \u003cp\u003eOptical Filter Configurations in Fluorescence Instruments 52\u003c\/p\u003e \u003cp\u003eFluorescence Filters Impact Optical System Performance 63\u003c\/p\u003e \u003cp\u003eTunable Optical Filters 71\u003c\/p\u003e \u003cp\u003eConclusion 75\u003c\/p\u003e \u003cp\u003eLiterature Cited 76\u003c\/p\u003e \u003cp\u003eProper Alignment and Adjustment of the Light Microscope 77\u003c\/p\u003e \u003cp\u003eMajor Components of the Light Microscope 78\u003c\/p\u003e \u003cp\u003eBasic Imaging and K¨ohler Illumination Light Paths for Bright-Field, Fluorescence, and Dark-Field Microscopy 83\u003c\/p\u003e \u003cp\u003eBasic Imaging for Dark-Field Microscopy 85\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Alignment for K¨ohler Illumination in Bright-Field, Transmitted Light Microscopy 86\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Alignment of the Eyepieces 89\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Alignment for K¨ohler Illumination in Epifluorescence Microscopy 90\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Alignment for Phase-Contrast Microscopy 92\u003c\/p\u003e \u003cp\u003eBasic Protocol 5: Alignment for DIC Microscopy 94\u003c\/p\u003e \u003cp\u003eAlignment for Dark-Field Microscopy 98\u003c\/p\u003e \u003cp\u003eBasic Protocol 6: Alignment for Low-Power Magnification Dark-Field Microscopy 99\u003c\/p\u003e \u003cp\u003eBasic Protocol 7: Alignment for High-Power Magnification Dark-Field Illumination 100\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: Matching Microscope Magnification to Detector Resolution 101\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Calibrating Image Magnification with a Stage Micrometer 102\u003c\/p\u003e \u003cp\u003eTests for the Optical Performance of the Microscope 103\u003c\/p\u003e \u003cp\u003eSupport Protocol 3: Testing Phase-Contrast and DIC Using Diatom Testing Slide 103\u003c\/p\u003e \u003cp\u003eSupport Protocol 4: Testing Phase-Contrast, Dark-Field, and DIC Microscopes Using a Squamous Cheek Cell Test Slide 103\u003c\/p\u003e \u003cp\u003eSupport Protocol 5: Testing Fluorescence Using a Red, Green, and Blue Fluorescent Tissue Culture Cell Test Slide 103\u003c\/p\u003e \u003cp\u003eSupport Protocol 6: Care and Cleaning of Microscope Optics 105\u003c\/p\u003e \u003cp\u003eCommentary 106\u003c\/p\u003e \u003cp\u003eLiterature Cited 107\u003c\/p\u003e \u003cp\u003e\u003cb\u003eChapter 2 Basic Methods\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eIntroduction 111\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection I Sample Preparation for Conventional Microscopy\u003c\/b\u003e\u003cbr\u003e \u003cbr\u003e Cryosectioning 113\u003c\/p\u003e \u003cp\u003eBasic Protocol: Specimen Preparation and Sectioning 113\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: Tissue Fixation and Sucrose Infusion 117\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Perfusion of Adult Mice 117\u003c\/p\u003e \u003cp\u003eReagents and Solutions 118\u003c\/p\u003e \u003cp\u003eCommentary 119\u003c\/p\u003e \u003cp\u003eLiterature Cited 120\u003c\/p\u003e \u003cp\u003eImmunohistochemistry 121\u003c\/p\u003e \u003cp\u003eIntroduction 121\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Immunofluorescent Labeling of Cells Grown as Monolayers 121\u003c\/p\u003e \u003cp\u003eAlternate Protocol 1: Immunofluorescent Labeling of Suspension Cells 123\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Immunofluorescent Labeling of Tissue Sections 124\u003c\/p\u003e \u003cp\u003eAlternate Protocol 2: Immunofluorescent Labeling Using Streptavidin-Biotin Conjugates 125\u003c\/p\u003e \u003cp\u003eAlternate Protocol 3: Immunofluorescent Double-Labeling of Tissue Sections 126\u003c\/p\u003e \u003cp\u003eReagents and Solutions 127\u003c\/p\u003e \u003cp\u003eCommentary 127\u003c\/p\u003e \u003cp\u003eLiterature Cited 131\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection II Dyes and Probes\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eA Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures 133\u003c\/p\u003e \u003cp\u003eIntroduction 133\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: BacMam Constructs 136\u003c\/p\u003e \u003cp\u003eAlternate Protocol 1: Non-Pseudo-Typed BacMam Viruses\/Hard-To-Transduce Cell Types 140\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Actin Labeling 141\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Autophagosome Labeling by Transduction of Cells with Premo Autophagy Sensor GFP-LC3B 142\u003c\/p\u003e \u003cp\u003eAlternate Protocol 2: Performing Autophagosome Labeling with an Antibody 143\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Wheat Germ Agglutinin Conjugates for Plasma Membrane Labeling 145\u003c\/p\u003e \u003cp\u003eBasic Protocol 5: Endoplasmic Reticulum and Nuclear Membrane Labeling Using ER-Tracker Reagents 145\u003c\/p\u003e \u003cp\u003eBasic Protocol 6: Labeling Endosomes with pHrodo 10k Dextran 146\u003c\/p\u003e \u003cp\u003eBasic Protocol 7: Labeling Golgi Apparatus Using Dye-Labeled Ceramides 147\u003c\/p\u003e \u003cp\u003eBasic Protocol 8: Labeling Lysosomes Using LysoTracker Red DND-99 149\u003c\/p\u003e \u003cp\u003eBasic Protocol 9: Labeling Mitochondria Using MitoTracker Red CMXRos 150\u003c\/p\u003e \u003cp\u003eBasic Protocol 10: Labeling Nucleoli Using SYTO RNASelect Green 152\u003c\/p\u003e \u003cp\u003eBasic Protocol 11: Labeling Peroxisomes Using CellLight BacMam 2.0 Peroxisomes-GFP 153\u003c\/p\u003e \u003cp\u003eAlternate Protocol 3: Labeling Peroxisomes Using Antibodies 154\u003c\/p\u003e \u003cp\u003eBasic Protocol 12: Labeling Tubulin Microtubules with TubulinTracker Green 156\u003c\/p\u003e \u003cp\u003eBasic Protocol 13: Labeling Whole Cells or Cytoplasm with 5(6)-CFDA SE 156\u003c\/p\u003e \u003cp\u003eReagents and Solutions 158\u003c\/p\u003e \u003cp\u003eCommentary 161\u003c\/p\u003e \u003cp\u003eLiterature Cited 197\u003c\/p\u003e \u003cp\u003eInternet Resources 203\u003c\/p\u003e \u003cp\u003eThe Fluorescent Protein Color Palette 207\u003c\/p\u003e \u003cp\u003eIntroduction 207\u003c\/p\u003e \u003cp\u003eFluorescent Protein Brightness and Maturation 210\u003c\/p\u003e \u003cp\u003ePhototoxicity and Photostability 212\u003c\/p\u003e \u003cp\u003eOligomerization 214\u003c\/p\u003e \u003cp\u003eThe Fluorescent Protein Color Palette 216\u003c\/p\u003e \u003cp\u003eOptical Highlighter Fluorescent Proteins 232\u003c\/p\u003e \u003cp\u003eThe Future of Fluorescent Proteins 239\u003c\/p\u003e \u003cp\u003eLiterature Cited 239\u003c\/p\u003e \u003cp\u003ePhotoactivation and Imaging of Optical Highlighter Fluorescent Proteins 247\u003c\/p\u003e \u003cp\u003eIntroduction 247\u003c\/p\u003e \u003cp\u003eBackground 247\u003c\/p\u003e \u003cp\u003eRequirements for Highlighting Fluorescent Proteins 252\u003c\/p\u003e \u003cp\u003eOptimization Procedures 253\u003c\/p\u003e \u003cp\u003eGeneral Photoactivation Experiment 255\u003c\/p\u003e \u003cp\u003eUses of Optical Highlighter Fluorescent Proteins 256\u003c\/p\u003e \u003cp\u003eApplication of Optical Highlighter Fluorescent Proteins in Cytometry 258\u003c\/p\u003e \u003cp\u003eFuture Directions of Optical Highlighter Fluorescent Proteins 258\u003c\/p\u003e \u003cp\u003eAcknowledgement 259\u003c\/p\u003e \u003cp\u003eLiterature Cited 259\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection III Optical Sectioning Microscopy\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eBasic Confocal Microscopy 261\u003c\/p\u003e \u003cp\u003eIntroduction 261\u003c\/p\u003e \u003cp\u003eBasis of Optical Sectioning 263\u003c\/p\u003e \u003cp\u003eConfiguration of an LSCM 265\u003c\/p\u003e \u003cp\u003ePractical Guidelines 268\u003c\/p\u003e \u003cp\u003eCommentary 275\u003c\/p\u003e \u003cp\u003eAcknowledgements 278\u003c\/p\u003e \u003cp\u003eLiterature Cited 278\u003c\/p\u003e \u003cp\u003eKey References 280\u003c\/p\u003e \u003cp\u003eInternet Resources 280\u003c\/p\u003e \u003cp\u003eEvaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous Factors to Consider 283\u003c\/p\u003e \u003cp\u003eIntroduction 283\u003c\/p\u003e \u003cp\u003eApplications 285\u003c\/p\u003e \u003cp\u003eHardware 286\u003c\/p\u003e \u003cp\u003eSoftware 288\u003c\/p\u003e \u003cp\u003eQuality Assurance (QA) 289\u003c\/p\u003e \u003cp\u003eService, Support, and Company 293\u003c\/p\u003e \u003cp\u003eMaintenance\/Cleanup Protocol 294\u003c\/p\u003e \u003cp\u003ePrice 294\u003c\/p\u003e \u003cp\u003eRecommendation from Colleagues 294\u003c\/p\u003e \u003cp\u003eSummary and Conclusions 294\u003c\/p\u003e \u003cp\u003eDisclaimer 295\u003c\/p\u003e \u003cp\u003eResources Listed 295\u003c\/p\u003e \u003cp\u003eAcknowledgements 295\u003c\/p\u003e \u003cp\u003eLiterature Cited 295\u003c\/p\u003e \u003cp\u003e3D Deconvolution Microscopy 297\u003c\/p\u003e \u003cp\u003eIntroduction 297\u003c\/p\u003e \u003cp\u003eImage Formation 297\u003c\/p\u003e \u003cp\u003eResolution and Sampling 301\u003c\/p\u003e \u003cp\u003eEstimating and Optimizing the PSF 302\u003c\/p\u003e \u003cp\u003eDeblurring and Deconvolution Algorithms 303\u003c\/p\u003e \u003cp\u003eBlind Deconvolution 306\u003c\/p\u003e \u003cp\u003eExample Deconvolution Results 307\u003c\/p\u003e \u003cp\u003eDeconvolution Software 309\u003c\/p\u003e \u003cp\u003eBasic Protocol: Data Acquisition and Deconvolution Analysis 312\u003c\/p\u003e \u003cp\u003eConcluding Remarks 315\u003c\/p\u003e \u003cp\u003eLiterature Cited 315\u003c\/p\u003e \u003cp\u003eKey References 316\u003c\/p\u003e \u003cp\u003eInternet Resources 316\u003c\/p\u003e \u003cp\u003eMulti-Photon Imaging 317\u003c\/p\u003e \u003cp\u003eIntroduction 317\u003c\/p\u003e \u003cp\u003eMulti-Photon Microscopy 317\u003c\/p\u003e \u003cp\u003eMulti-Photon Imaging in Practice 323\u003c\/p\u003e \u003cp\u003eConcluding Remarks 328\u003c\/p\u003e \u003cp\u003eLiterature Cited 328\u003c\/p\u003e \u003cp\u003e\u003cb\u003eChapter 3 Applications\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eIntroduction 333\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection I Basic Live Cell Imaging\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eBuilding a Live-Cell Microscope: What You Need and How to Do It 335\u003c\/p\u003e \u003cp\u003eDefining the System 335\u003c\/p\u003e \u003cp\u003eBuilding a Live-Cell Scope: Components and Considerations 337\u003c\/p\u003e \u003cp\u003eTransmitted Light Choices 344\u003c\/p\u003e \u003cp\u003eSummary 346\u003c\/p\u003e \u003cp\u003eTime-Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single-Cell Level 347\u003c\/p\u003e \u003cp\u003eIntroduction 347\u003c\/p\u003e \u003cp\u003eSystem Setup 348\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Time-Lapse Acquisition Using Adherent Cells 349\u003c\/p\u003e \u003cp\u003eAlternate Protocol 1: Time-Lapse Acquisition with Endpoint Assay to Mark S-Phase Cells 350\u003c\/p\u003e \u003cp\u003eAlternate Protocol 2: Time-Lapse Acquisition Using Suspension Cells 351\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Sequence Analysis for Mitosis Event or Cell Death 352\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Data Mining—Normalized Event Distribution 353\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Data Mining—Time-to-Event Curves 354\u003c\/p\u003e \u003cp\u003eBasic Protocol 5: Data Mining—Duration of Mitotic Event 355\u003c\/p\u003e \u003cp\u003eBasic Protocol 6: Data Mining—G2 Checkpoint Breaching 355\u003c\/p\u003e \u003cp\u003eBasic Protocol 7: Data Mining—Deriving Basic Lineage Parameters 356\u003c\/p\u003e \u003cp\u003eCommentary 356\u003c\/p\u003e \u003cp\u003eLiterature Cited 359\u003c\/p\u003e \u003cp\u003eInternet Resources 360\u003c\/p\u003e \u003cp\u003eAnalysis of Mitochondrial Dynamics and Functions Using Imaging Approaches 361\u003c\/p\u003e \u003cp\u003eIntroduction 361\u003c\/p\u003e \u003cp\u003eStrategic Planning 361\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: High-Resolution z-Stack and Time-Lapse Imaging of Mitochondria 363\u003c\/p\u003e \u003cp\u003eAlternate Protocol: Imaging Mitochondrial Morphology Alterations 366\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Fluorescence Recovery After Photobleaching on Mitochondria 367\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Microirradiation Assay to Assess Electrical Continuity in Mitochondria 372\u003c\/p\u003e \u003cp\u003eSupport Protocol: Staining Mitochondria in Live Cells to Assess Mitochondrial Function by Imaging 375\u003c\/p\u003e \u003cp\u003eCommentary 378\u003c\/p\u003e \u003cp\u003eLiterature Cited 382\u003c\/p\u003e \u003cp\u003eAnalysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching (FRAP) 385\u003c\/p\u003e \u003cp\u003eIntroduction 385\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: How to Set Up a FRAP Experiment 387\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Confocal FRAP Measurements of the Lateral Diffusion of Plasma Membrane Proteins and Lipids 391\u003c\/p\u003e \u003cp\u003eAlternate Protocol 1: Lateral Diffusion Measurements for a Rapidly Diffusing Soluble Protein 393\u003c\/p\u003e \u003cp\u003eAlternate Protocol 2: FRAP Analysis of Intracellular Trafficking Kinetics 395\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Working with FRAP Data 397\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Further Analysis of FRAP Data to Obtain Diffusion Coefficients 399\u003c\/p\u003e \u003cp\u003eCommentary 401\u003c\/p\u003e \u003cp\u003eAcknowledgements 411\u003c\/p\u003e \u003cp\u003eLiterature Cited 411\u003c\/p\u003e \u003cp\u003eConfocal Imaging of Cell Division 415\u003c\/p\u003e \u003cp\u003eIntroduction 415\u003c\/p\u003e \u003cp\u003eSpinning Disk Confocal 415\u003c\/p\u003e \u003cp\u003eConfocal Imaging of Chromosome Condensation in C. elegans Embryos 420\u003c\/p\u003e \u003cp\u003eConfocal Imaging of Spindle Assembly and Chromosome Dynamics 421\u003c\/p\u003e \u003cp\u003eConfocal Imaging of Cytokinesis 424\u003c\/p\u003e \u003cp\u003eDiscussion 425\u003c\/p\u003e \u003cp\u003eAcknowledgements 426\u003c\/p\u003e \u003cp\u003eLiterature Cited 426\u003c\/p\u003e \u003cp\u003eTotal Internal Reflection Fluorescence (TIRF) Microscopy 429\u003c\/p\u003e \u003cp\u003eIntroduction 429\u003c\/p\u003e \u003cp\u003eThe Theory Behind the Technique 430\u003c\/p\u003e \u003cp\u003eTIRF Objectives 432\u003c\/p\u003e \u003cp\u003eEmpirically Determining Incident Angle\/Penetration Depth 434\u003c\/p\u003e \u003cp\u003eTIRF Imaging of Plasma Membrane Receptors in Neurons 436\u003c\/p\u003e \u003cp\u003eMulti-Wavelength TIRFM 438\u003c\/p\u003e \u003cp\u003eFinal Experimental Suggestions 441\u003c\/p\u003e \u003cp\u003eConcluding Remarks 442\u003c\/p\u003e \u003cp\u003eLiterature Cited 442\u003c\/p\u003e \u003cp\u003eTotal Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events 445\u003c\/p\u003e \u003cp\u003eIntroduction 445\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Through-the-Objective TIRF Protocol 445\u003c\/p\u003e \u003cp\u003eAlternate Protocol: Improved Uniformity in the Excitation Field Protocol 450\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Through-the-Prism TIRF Protocol 452\u003c\/p\u003e \u003cp\u003eCommentary 454\u003c\/p\u003e \u003cp\u003eLiterature Cited 465\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection II Fluorescence Resonance Energy Transfer\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eImaging Protein-Protein Interactions by F¨orster Resonance Energy Transfer (FRET) Microscopy in Live Cells 467\u003c\/p\u003e \u003cp\u003eCommentary 474\u003c\/p\u003e \u003cp\u003eLiterature Cited 479\u003c\/p\u003e \u003cp\u003eImaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy 481\u003c\/p\u003e \u003cp\u003eBasic Protocol: FRET Microscopy of Fixed Cells 482\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: Nuclear and Cytosolic Microinjection 485\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Protein Labeling with Cy3 487\u003c\/p\u003e \u003cp\u003eReagents and Solutions 490\u003c\/p\u003e \u003cp\u003eCommentary 490\u003c\/p\u003e \u003cp\u003eLiterature Cited 496\u003c\/p\u003e \u003cp\u003eUse of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide–Based Signaling Events in Isolated Perfused Lung 499\u003c\/p\u003e \u003cp\u003eIntroduction 499\u003c\/p\u003e \u003cp\u003eStrategic Planning 499\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Isolating and Perfusing Mouse Lung 500\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: No-Induced Protein Modifications Detected by FRET Using Spectral Confocal Microscopy 503\u003c\/p\u003e \u003cp\u003eReagents and Solutions 506\u003c\/p\u003e \u003cp\u003eCommentary 507\u003c\/p\u003e \u003cp\u003eLiterature Cited 510\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection III Imaging of Model Systems\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eFluorescence Imaging Techniques for Studying Drosophila Embryo Development 513\u003c\/p\u003e \u003cp\u003eIntroduction 513\u003c\/p\u003e \u003cp\u003eStrategic Planning 514\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Generation of Transgenic Drosophila for Live Fluorescence Microscopy Using the Gal4\/UAS System 525\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Preparation of Drosophila Embryos for Fluorescence Microscopy 529\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Time-Lapse Confocal Imaging of Living Drosophila Embryos 531\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Time-Lapse Imaging of Living Drosophila Embryos with Two-Photon Laser Scanning Microscopy 537\u003c\/p\u003e \u003cp\u003eBasic Protocol 5: Fluorescence Recovery After Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope\u003c\/p\u003e \u003cp\u003eCapable of Selective Photobleaching 540\u003c\/p\u003e \u003cp\u003eBasic Protocol 6: Fluorescence Loss in Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching 546\u003c\/p\u003e \u003cp\u003eBasic Protocol 7: Photoactivation in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching 548\u003c\/p\u003e \u003cp\u003eReagents and Solutions 553\u003c\/p\u003e \u003cp\u003eCommentary 553\u003c\/p\u003e \u003cp\u003eLiterature Cited 557\u003c\/p\u003e \u003cp\u003eTime-Lapse Imaging of Embryonic Neural Stem Cell Division in Drosophila by Two-Photon Microscopy 561\u003c\/p\u003e \u003cp\u003eIntroduction 561\u003c\/p\u003e \u003cp\u003eBasic Protocol: Time-Lapse Imaging by Two-Photon Microscopy 561\u003c\/p\u003e \u003cp\u003eSupport Protocol: Embryo Preparation 564\u003c\/p\u003e \u003cp\u003eCommentary 565\u003c\/p\u003e \u003cp\u003eAcknowledgements 569\u003c\/p\u003e \u003cp\u003eLiterature Cited 569\u003c\/p\u003e \u003cp\u003eImaging Tumor Cell Movement In Vivo 571\u003c\/p\u003e \u003cp\u003eIntroduction 571\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Generation and In Vivo Imaging of Mammary Tumors 571\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: In Vivo Imaging Microscope Setup 579\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Labeling Vasculature and Macrophages 580\u003c\/p\u003e \u003cp\u003eSupport Protocol 3: Blood Vessel Imaging Using an Indwelling Catheter 581\u003c\/p\u003e \u003cp\u003eSupport Protocol 4: Second Harmonic Fiber Imaging 583\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Multiphoton Time-Lapse Image Analysis Using ImageJ and Custom Plugins 583\u003c\/p\u003e \u003cp\u003eSupport Protocol 5: Separation of Spectral Overlap 586\u003c\/p\u003e \u003cp\u003eReagents and Solutions 587\u003c\/p\u003e \u003cp\u003eCommentary 587\u003c\/p\u003e \u003cp\u003eLiterature Cited 589\u003c\/p\u003e \u003cp\u003eLive-Animal Imaging of Renal Function by Multiphoton Microscopy 591\u003c\/p\u003e \u003cp\u003eIntroduction 591\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Glomerular Permeability 592\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Proximal Tubule Endocytosis 593\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Vascular Flow 594\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Vascular Permeability 596\u003c\/p\u003e \u003cp\u003eBasic Protocol 5: Mitochondrial Function 597\u003c\/p\u003e \u003cp\u003eBasic Protocol 6: Apoptosis 598\u003c\/p\u003e \u003cp\u003eSupport Protocol: Anesthesia and Surgical Creation of a Retroperitoneal Surgical Window for Intravital Imaging 599\u003c\/p\u003e \u003cp\u003eReagents and Solutions 602\u003c\/p\u003e \u003cp\u003eCommentary 602\u003c\/p\u003e \u003cp\u003eLiterature Cited 608\u003c\/p\u003e \u003cp\u003eBiological Second and Third Harmonic Generation Microscopy 611\u003c\/p\u003e \u003cp\u003eStrategic Planning 612\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Designing a Microscope System for HHGM 612\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Detection of Fibrillar Collagen in Connective Tissue Ex Vivo 619\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Detection of SHG in Mouse Tissues by Intravital Microscopy 621\u003c\/p\u003e \u003cp\u003eBasic Protocol 4: Simultaneous Detection of Cells and Collagen Fibers In Vitro and In Vivo 622\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: Cytoplasmic Staining of Live Cells 625\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Establishment of 3-D Collagen Cultures 625\u003c\/p\u003e \u003cp\u003eReagents and Solutions 626\u003c\/p\u003e \u003cp\u003eCommentary 626\u003c\/p\u003e \u003cp\u003eAcknowledgements 632\u003c\/p\u003e \u003cp\u003eLiterature Cited 632\u003c\/p\u003e \u003cp\u003eTwo-Photon Imaging of the Immune System 635\u003c\/p\u003e \u003cp\u003eIntroduction 635\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Preparing the Thymus of a Mouse for Two-Photon Imaging 636\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Preparing the Mesenteric Lymph Nodes (MLNs) of a Mouse for Two-Photon Imaging 637\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Preparing Segments from the Intestine of a Mouse for Two-Photon Imaging 639\u003c\/p\u003e \u003cp\u003eAlternate Protocol 1: Agarose Embedding of a Small Tissue Sample or Organotypic Cultures 640\u003c\/p\u003e \u003cp\u003eAlternate Protocol 2: Preparing Thymic Slices for Two-Photon Imaging 642\u003c\/p\u003e \u003cp\u003eAlternate Protocol 3: Overlaying Thymic Slices with Fluorescently Labeled Cells 645\u003c\/p\u003e \u003cp\u003eSupport Protocol: Setting Up Two-Photon Imaging Conditions 646\u003c\/p\u003e \u003cp\u003eReagents and Solutions 647\u003c\/p\u003e \u003cp\u003eCommentary 647\u003c\/p\u003e \u003cp\u003eLiterature Cited 654\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection IV Super-Resolution Methods\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eSuper-Resolution Microscopy: A Comparative Treatment 657\u003c\/p\u003e \u003cp\u003eIntroduction 657\u003c\/p\u003e \u003cp\u003eSuper-Resolution Imaging Methodologies 657\u003c\/p\u003e \u003cp\u003ePoint-Spread Function Engineering 668\u003c\/p\u003e \u003cp\u003eConcluding Remarks 677\u003c\/p\u003e \u003cp\u003eAcknowledgements 677\u003c\/p\u003e \u003cp\u003eLiterature Cited 677\u003c\/p\u003e \u003cp\u003ePhotoactivated Localization Microscopy (PALM) of Adhesion Complexes 683\u003c\/p\u003e \u003cp\u003eIntroduction 683\u003c\/p\u003e \u003cp\u003eStrategic Planning 683\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Preparing PALM Instrumentation 687\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: PALM Imaging tdEos\/Paxillin Distributions in Fixed Cells 697\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Dual-Color PALM Imaging of tdEos\/Vinculin and Dronpa α-Actinin in Fixed Cells 701\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: Preparing Clean Coverslips 704\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Transfection of tdEos\/Paxillin into HFF-1 Cells 705\u003c\/p\u003e \u003cp\u003eReagents and Solutions 707\u003c\/p\u003e \u003cp\u003eCommentary 708\u003c\/p\u003e \u003cp\u003eLiterature Cited 710\u003c\/p\u003e \u003cp\u003eComparative and Practical Aspects of Localization-Based Super-Resolution Imaging 713\u003c\/p\u003e \u003cp\u003eIntroduction 713\u003c\/p\u003e \u003cp\u003eBasic Protocol 1: Multi-Channel Labeling of Microtubules and Mitochondria with STORM Tandem Dye Pairs 713\u003c\/p\u003e \u003cp\u003eSupport Protocol 1: Dye Preparation and Secondary Antibody Labeling 715\u003c\/p\u003e \u003cp\u003eBasic Protocol 2: Buffer and Imaging Conditions for Synthetic Photoswitchable Dyes 716\u003c\/p\u003e \u003cp\u003eBasic Protocol 3: Labeling Proteins via SNAP Tags for Live-Cell Localization Super Resolution 717\u003c\/p\u003e \u003cp\u003eSupport Protocol 2: Buffer and Imaging Conditions for Live-Cell Localization Super Resolution 719\u003c\/p\u003e \u003cp\u003eCommentary 719\u003c\/p\u003e \u003cp\u003eAcknowledgements 723\u003c\/p\u003e \u003cp\u003eLiterature Cited 723\u003c\/p\u003e \u003cp\u003e\u003cb\u003eChapter 4 Image Processing\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eIntroduction 727\u003c\/p\u003e \u003cp\u003eEthical Considerations When Altering Digital Images 729\u003c\/p\u003e \u003cp\u003eIntroduction 729\u003c\/p\u003e \u003cp\u003eGolden Rules 729\u003c\/p\u003e \u003cp\u003eGuidelines from Specific Journals 731\u003c\/p\u003e \u003cp\u003eLiterature Cited 733\u003c\/p\u003e \u003cp\u003eFrom Image to Data Using Common Image-Processing Techniques 735\u003c\/p\u003e \u003cp\u003eIntroduction 735\u003c\/p\u003e \u003cp\u003eImage Anatomy 735\u003c\/p\u003e \u003cp\u003eImage Processing 736\u003c\/p\u003e \u003cp\u003eConcluding Remarks 751\u003c\/p\u003e \u003cp\u003eLiterature Cited 751\u003c\/p\u003e \u003cp\u003ePractical Considerations When Altering Digital Images 753\u003c\/p\u003e \u003cp\u003eIntroduction 753\u003c\/p\u003e \u003cp\u003eSampling Resolution 753\u003c\/p\u003e \u003cp\u003eResampling 755\u003c\/p\u003e \u003cp\u003eAcquiring Images 758\u003c\/p\u003e \u003cp\u003ePhotoshop and Scientific Image–Analysis Programs 762\u003c\/p\u003e \u003cp\u003eOptimizing the Display 764\u003c\/p\u003e \u003cp\u003eUsing Images from Vector Programs and PowerPoint 765\u003c\/p\u003e \u003cp\u003eAltering Images Using Photoshop 766\u003c\/p\u003e \u003cp\u003eInserting Files into PowerPoint 784\u003c\/p\u003e \u003cp\u003eLiterature Cited 785\u003c\/p\u003e \u003cp\u003eAppendix 1: Common Stock Solutions, Buffers, and Media 787\u003c\/p\u003e \u003cp\u003eIndex 791\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSimon C. Watkins\u003c\/b\u003e, Ph.D.: Dr. Watkins is a Professor and Vice Chair of the Cell Biology and Physiology Department at the University of Pittsburgh, Pennsylvania, and holds a secondary appointment as Professor in the Department of Immunology at the same institution. He is also the Founder and Director of the Center for Biologic Imaging at the University of Pittsburgh. Dr. Watkins is an expert in microscopy and imaging and is an Editor for \u003ci\u003eCurrent Protocols in Cytometry\u003c\/i\u003e where he is in charge of the chapters on 'Image Cytometry Instrumentation' and 'Cellular and Molecular Imaging'.\u003c\/p\u003e \u003cp\u003e\u003cb\u003eClaudette M. St. Croix\u003c\/b\u003e, Ph.D.: Dr. St. Croix is an Assistant Professor in the Department of Environmental and Occupational Health at the University of Pittsburgh, Pennsylvania, and holds a secondary appointment as Assistant Professor in the Cell Biology and Physiology Department at the same institution. She is also Assistant Director of the Center for Biologic Imaging at the University of Pittsburgh. Dr. St Croix's highly inventive applications of advanced imaging technologies have led to numerous invitations to present her work nationally and internationally and to take lead roles in well respected courses such as Quantitative Fluorescence Microscopy (run at Mount Desert Island Biology Laboratory) and at the national Microscopy Society of America annual meeting. She is also an author for \u003ci\u003eCurrent Protocols in Cytometry\u003c\/i\u003e.\u003c\/p\u003e  \u003cp\u003eThe 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.\u003c\/p\u003e \u003cp\u003eThis 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 \u003ci\u003eCurrent Protocols\u003c\/i\u003e 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.\u003c\/p\u003e \u003cul\u003e \u003cli\u003eFor graduate students, postdocs, and individuals who are branching into new areas as an entry into the specialized techniques of microscopy\u003c\/li\u003e \u003cli\u003eA resource that encourages the use of the microscope beyond that of the simple light microscope\u003c\/li\u003e \u003cli\u003eAn essential reference for core facilities at academic institutions\u003c\/li\u003e \u003cli\u003eAn \"instructional\" text essential for each of these core resources\u003c\/li\u003e \u003c\/ul\u003e","brand":"Current Protocols","offers":[{"title":"Default Title","offer_id":47989016297701,"sku":"NP9781118044315","price":173.95,"currency_code":"USD","in_stock":false}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/1842\/7735\/files\/9781118044315.jpg?v=1761782448","url":"https:\/\/k12savings.com\/es\/products\/current-protocols-select-isbn-9781118044315","provider":"K12savings","version":"1.0","type":"link"}