Holdings: Handbook of biological confocal microscopy

Handbook of biological confocal microscopy:

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Bibliographic Details
Format:	Book
Language:	English
Published:	New York, NY Springer 2006
Edition:	3. ed.
Subjects:	Microscopia (métodos;técnicas) Microscopie confocale Terceira dimensão Confocal microscopy Imaging, Three-Dimensional Microscopy, Confocal Biowissenschaften Konfokale Mikroskopie Biologie Mikroskopie Konferenzschrift > 1989 > San Antonio Tex.
Online Access:	Inhaltsverzeichnis
Physical Description:	XXVIII, 985 S. zahlr. Ill., graph. Darst.
ISBN:	038725921X 9780387259215

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adam_text	Contents Preface to the Third Edition Preface to the Second Edition Contributors CHAPTER CONFOCAL SCANNED IMAGING IN LIGHT MICROSCOPY Shinya Light Microscopy Lateral Resolution Axial Resolution Depth of Field Confocal Imaging Impact of Video Nipkow Disk Electron-Beam-Scanning Television Impact of Modern Video Lasers and Microscopy Holography Laser Illumination Laser-Illuminated Confocal Microscopes Confocal Laser-Scanning Microscope Two- and Multi-Photon Microscopy Is Laser-Scanning Confocal Microsopy a Cure-Ail? Speed of Image or Data Acquisition Yokogawa Disk-Scanning Confocal System Depth of Field in Phase-Dependent Imaging Other Optical and Mechanical Factors Affecting Confocal Microscopy Lens Aberration Unintentional Beam Deviation Contrast Transfer and Resolution in Confocal Versus Non-Confocal Microscopy Summary CHAPTER CONFOCAL MICROSCOPY James B. Pawley Introduction What Limits? Counting Statistics: The Importance of Source Brightness Specimen Response: Dye Saturation A Typical Problem Practical Photon Efficiency Losses in the Optical System Detection and Measurement Losses Where Have All the Photons Gone? Resolution: How Much Is Enough? Can Resolution Be Too High? Limitations Imposed by Spatial and Temporal Quantization Practical Considerations Relating Resolution to Distortion Conclusion CHAPTER Jens Rietdorf Introduction Regulating the Intensity Wavelength Selective Filtering Devices Selecting the Wavelength of the Illumination and the Detected Light Separating the Light Paths Conventional Filters Interference Filters Dichroic and Polarizing Beam-Splitters Filters and Dispersive Elements for Multi-Channel Detection Mechanical Scanners Galvanometer Scanners General Specifications Acousto-Optical Components Acousto-Optical Deflectors Acousto-Optical Modulators Acousto-Optical Tunable Filters Acousto-Optical Beam-Splitters Electro-Optical Modulators Piezoelectric Scanners Polarizing Elements Removing Excess Light CHAPTER LEVELS: DIGITIZING IMAGE DATA James B. Pawley Contrast Transfer Function, Points, and Pixels Pixels, Images, and the Contrast Transfer Function Digitization and Pixels Digitization of Images How Big Should a Pixel Be? Sampling and Quantum Noise The Nyquist Criterion Estimating the Expected Resolution of an Image The Story So Far Reality Check? Is Over-Sampling Ever Wise? Under-Sampling? Digitizing Trade-Offs xii Contents Nyquist Some Gray Levels, "Noise," and Photodetector Performance Optical Density The Zone System: Quantified Photography Linearity: Do We Need It? Gray Levels in Images Recorded Using Charge-Coupled Devices: The Intensity Spread Function What Counts as Noise? Measuring the Intensity Spread Function Calibrating a Charge-Coupled Device to Measure the ISF "Fixed-Pattern" Noise Gain-Register Charge-Coupled Devices Multiplicative Noise Trade-Offs CHAPTER CONFOCAL MICROSCOPY Enrico Gratton and Martin J. vandeVen Introduction Laser Power Requirements The Basic Laser Principle of Operation Pumping Power Requirements Laser Modes: Longitudinal (Axial) and Transverse Polarization Coherent Properties of Laser Light Phase Randomization: Scrambling the Coherence Properties of Laser Light Measures to Reduce the Coherence Length of Laser Light Heat Removal Other Installation Requirements Attenuation of Laser Beams Stabilization of Intensity, Wavelength, and Beam Position in Lasers Sources of Noise in Lasers Spatial Beam Characteristics Laser Requirements for Biological Confocal Laser Scanning Microscopy-Related Techniques Optical Tweezers Total Internal Reflection Microscopy Confocal Raman Confocal Laser Scanning Microscopy for Chemical Imaging Non-Linear Confocal Microscopy Nanosurgery and Types of Lasers Continuous Wave Lasers Gas Lasers Dye Lasers Solid-State Lasers Thin Disk Lasers Pulsed Lasers Classification of Pulsed Laser Systems Nitrogen Lasers Excimer Lasers Metal Vapor Lasers Dye Lasers Modulated Diode Lasers Diode Pumped Solid State Laser in Pulsed Mode Ultrafast Diode Pumped Solid State Lasers Titanium-Sapphire and Related Ultrafast Lasers White Light Continuum Lasers Ultrafast Fiber Lasers Wavelength Expansion Through Non-Linear Techniques Second and Higher Harmonic Generation: SHG, THG, FHG Label-Free Microscopy Sum or Difference Mixing Optical Parametric Oscillators and Optical Parametric Amplifiers Pulse Length Measurement Maintenance Maintenance of Active Laser Media Maintenance of Pumping Media Maintenance of the Optical Resonator Maintenance of Other System Components Troubleshooting Safety Precautions Beam Stops Curtains Laser Goggles Screens Exposure Effects, Warning Signs, and Interlocks Infrared Paper Conclusion CHAPTER FOR THREE-DIMENSIONAL MICROSCOPY Andreas Nolte, James B. Pawley, and Introduction General Remarks on Choice of Excitation Light Sources Scrambling and Filtering the Light Types of Sources and Their Features Structure Wavelength Stability in Time and Wavelength Radiance Control Measuring What Comes Through the Illumination System The Bare Minimum Types of Confocal Microscopes That Can Use Non-Laser Light Sources Tandem Scanning: Basic Description Single-Sided Disk Scanning: Basic Description Exposure Time and Source Brightness Future Trends CHAPTER CONFOCAL MICROSCOPY H. Ernst Keller Introduction Aberrations of Refractive Systems Defocusing Monochromatic Aberrations Chromatic Aberrations Contents xiii Finite Working Distance Optical Materials Anti-Reflection Coatings Transmission of Microscope Objectives Conclusion CHAPTER IN OPTICAL MICROSCOPY Ping-Chin Cheng Introduction Sources of Contrast Absorption Contrast Scattering and Reflection Contrast Phase Contrast Fluorescence Contrast Contrast Related to Excitation Wavelength Change Negative Contrast Special Concerns in Ultraviolet and Near-Infrared Range Confocal Microscopy Total Internal Reflection Contrast Harmonic Generation Contrast Geometric Contrast z-Contrast in Confocal Microscopy Total Internal Refraction Fluorescence Contrast Fluorescence Resonant Energy Transfer Fluorescence Recovery After Photobleaching (FRAP and FLIP) Structural Contrast Harmonic Generation Contrast Birefringence Contrast Derived Contrast (Synthetic Contrast) Ratiometric Deconvolution Movement Contrast (Subtraction of Previous Image) Spectral Unmixing and Color Reassignment Effects of the Specimen: Spherical Aberration and Optical Heterogeneity Mounting Medium Selection Artificial Contrast Contrast Resulting from Instrument Vibration and Ambient Lighting Contrast Resulting from Interference of Cover Glass Surfaces Background Level and Ghost Images from the Transmission Illuminator Contrast Resulting from Differences in Photobleaching Dynamics Effect of Spectral Leakage and Signal Imbalance Between Different Channels New Contrasts: Fluorescence Lifetime and Coherent Antistokes Raman Spectroscopy Summary CHAPTER SYSTEM OF LASER-SCANNING CONFOCAL MICROSCOPES Ernst H.K. Introduction Design Principles Overview Telecentricity The Scanning System The Back-Focal Planes Practical Requirements Diffraction Limit Geometric Distortion Evaluation of the Illumination and Detection Systems Influence of Optical Elements Errors Evaluation of Optical Arrangements Evaluation of Scanner Arrangements Scanners Attachment to Microscopes Merit Functions Multi-Fluorescence Special Setups Setups for Fluorescence Recovery After Photobleaching Experiments Setups for Fluorescence Resonance Energy Transfer Experiments Setups for the Integration of Optical Tweezers Setups for the Integration of Laser Cutters Setups for the Observation of Living Specimens Miniaturization and Computer Control Thermal Stability Vibration Isolation Conclusions and Future Prospects CHAPTER MICROSCOPY Derek Toomre and James B. Pawley Introduction Background Living Cell Imaging: Probing the Future A Need for Speed and Less Photobleaching Advantages and Limitations of Confocal Laser-Scanning Microscopes Other Imaging and Deconvolution Confocal Disk-Scanning Microscopy Nipkow Disk A Renaissance Confocal Imaging Disadvantages Critical Parameters in Pinhole and Slit Disks Fill Factor and Spacing Interval Lateral Resolution Pinhole/Slit Size Axial Resolution Types of Disk-Scanning General Considerations Disk Scanners for Backscattered Light Imaging CARV, Microscopes The Yokogawa Microlens Approach New Fast Slit Scanner New Detectors Image Intensifies On-Chip Electron Multiplying Charge-Coupled Device xiv Contents Electron Disk Scanners Applications and Examples of Confocal Disk-Scanning Microscopes Comparison with Epi-Fluorescence Imaging Fast 3D/4D Imaging Blazingly Fast Confocal Imaging Future Developments? Summary CHAPTER FUNCTION OF HIGH NUMERICAL APERTURE MICROSCOPE OBJECTIVE LENSES Rimas Introduction Measuring Point Spread Function Fiber-Optic Interferometer Point Spread Function Measurements Chromatic Aberrations Apparatus Axial Shift Pupil Function Phase-Shifting Zernike Polynomial Fit Restoration of a Empty Aperture Miscellanea Temperature Variations Polarization Effects Apodization Conclusion CHAPTER CONFOCAL MICROSCOPY Jonathan Art Introduction The Quantal Nature of Light Interaction of Photons with Materials Thermal Effects Direct Effects Photoconductivity Photovoltaic Photoemissive Comparison of Detectors Noise Internal to Detectors Noise in Internal Detectors Noise in Photoemissive Devices Statistics of Photon Flux and Detectors Representing the Pixel Value Conversion Techniques Assessment of Devices Point Detection Assessment and Optimization Field Detection Assessment and Optimization Detectors Present and Future CHAPTER METHODS Rainer Heintzmann Introduction 265 Experimental Considerations Pattern Generation Computing Optical Sections from Structured-Illumination Data Resolution Improvement by Structured Illumination Nonlinear Structured Illumination Summary CHAPTER MULTI-DIMENSIONAL MICROSCOPY IMAGES N.S. White Introduction Definitions What Is the Microscopist Trying to Achieve? Criteria for Choosing a Visualization System Why Do We Want to Visualize Multi-Dimensional Laser-Scanning Microscopy Data? Data and Dimensional Reduction Objective or Subjective Visualization? Prefiltering Identifying Unknown Structures Highlighting Previously Elucidated Structures Visualization for Multi-Dimensional Measurements What Confocal Laser Scanning Microscopy Images Can the Visualization System Handle? Image Data: How Are Image Values Represented in the Program? What Dimensions Can the Images and Views Have? Standard File Formats for Calibration and Interpretation How Will the System Generate the Reconstructed Views? Assessing the Four Basic Steps in the Generation of Reconstructed Views Loading the Image Subregion Choosing a View: The 5D Image Display Space Mapping the Image Space into the Display Space How Do z-Information? Mapping the Data Values into the Display How Can Intensities Be Used to Retain г Hidden-Object Removal Adding Realism to the View How Can I Make Measurements Using the Reconstructed Views? Conclusion CHAPTER DIMENSIONAL IMAGE ANALYSIS METHODS FOR CONFOCAL MICROSCOPY Badrinath Omar Al-Kofahi, Khalid Al-Kofahi, William Shain, Donald H. Szarowsk, and James Introduction Types of Automated Image Analysis Studies Contents xv Common Types of Biological Image Objects Specimen Preparation and Image Preprocessing Methods Data Collection Guidelines for Image Analysis Purposes Image Preprocessing Methods General Segmentation Methods Applicable to Confocal Data Bottom-Up Segmentation Methods Top-Down Segmentation Methods Hybrid Segmentation Methods Combining Bottom-Up and Top-Down Processing Example Illustrating Blob Segmentation Model-Based Object Merging Example Illustrating Segmentation of Tube-Like Objects Skeletonization Methods Vectorization Methods Example Combining Tube and Blob Segmentation Registration and Montage Synthesis Methods Methods for Quantitative Morphometry Methods for Validating the Segmentation and Making Corrections Analysis of Morphometric Data Discussion, Conclusion, and Future Directions CHAPTER MICROSCOPY: PHOTOPHYSICS AND PHOTOCHEMISTRY Roger Y. Tsien, Lauren Ernst, and Alan Waggoner Introduction Photophysical Problems Related to High Intensity Excitation Singlet State Saturation Triplet State Saturation Contaminating Background Signals What Is the Optimal Intensity? Photodestruction Specimens Dependency on Intensity or Its Time Integral? Strategies for Signal Optimization in the Face of Photobleaching Light Collection Efficiency Spatial Resolution Protective Agents Fluorophore Concentration Choice of Fluorophore Fluorescent Labels for Antibodies, Other Proteins, and Fluorescent Organic Dyes Phycobiliproteins DNA Luminescent Nanocrystals Fluorescent Lanthanide Chelates Fluorescent Indicators for Dynamic Intracellular Parameters Membrane Potentials Ion Concentrations pH Ca2+ Indicators Oxygen Sensor cAMP Indicators Fatty Acid Indicator Genetically Expressed Intracellular Fluorescent Indicators Green Fluorescent Protein Ligand-Binding Modules Ion Indicators Future Developments CHAPTER THE SELECTION AND APPLICATION OF FLUORESCENT PROBES Iain D. Johnson Introduction Selection Criteria for Dyes and Probes Organic Dyes Fluorescent Proteins: Green Fluorescent Protein and Phycobiliproteins Quantum Dots Multi-Photon Excitation Introducing the Probe to the Specimen Loading Methods Target Abundance and Considerations Interactions of Probes and Specimens Localization and Metabolism Perturbation and Cytotoxicity Under the Microscope Photobleaching Phototoxicity Summary CHAPTER SPECIMEN PRESERVATION FOR CONFOCAL FLUORESCENCE MICROSCOPY Robert Introduction Characteristics of Fixatives Glutaraldehyde Formaldehyde Fixation Staining and Mounting Methods Glutaraldehyde Fixation pH Immunofluorescence Staining Mounting the Specimen Critical Evaluation of Light Microscopy Fixation and Mounting Methods Use of the Cell Height to Evaluate the Fixation Method Use of Cell Height to Evaluate Mounting Media Well-Defined Structures Can Be Used to Evaluate Fixation Methods Comparison of In Vivo Labeled Cell Immunolabeled Cell General Notes Labeling Samples with Two or More Probes xvi Contents Triple Preparation of Tissue Specimens Labeling Thick Sections Refractive Index Mismatch Screening Antibodies on Glutaraldehyde-Fixed Specimens Microwave Fixation Conclusion CHAPTER OF LIVING CELLS Michael E. Dailey, Erik Manders, David R. Soil, and Mark Terasaki Introduction Overview of Living-Cell Confocal Imaging Techniques Time-Lapse Fluorescence Imaging Multi-Channel Time-Lapse Fluorescence Imaging Spectral Imaging and Linear Unmixing Fluorescence Recovery After Photobleaching Fluorescence Loss in Photobleaching Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Fluorescence Correlation Spectroscopy Fluorescence Photo-Uncaging/Photoactivation Optical Tweezers/Laser Trapping Physiological Fluorescence Imaging Combining Fluorescence and Other Imaging Modalities General Considerations for Confocal Microscopy of Living Cells Maintenance of Living Cells and Tissue Preparations Fluorescent Probes Minimizing Photodynamic Damage The Online Confocal Community A Convenient Test Specimen Specific Example I: Visualizing Chromatin Dynamics Using Very Low Light Levels Phototoxicity Reduction of Phototoxicity Improving Image Quality in Low-Dose Microscopy Low-Dose Imaging Conclusion Specific Example II: Multi-Dimensional Imaging of Microglial Cell Behaviors in Live Rodent Brain Slices Preparation of Central Nervous System Tissue Slices Fluorescent Staining Maintaining Tissue Health on the Microscope Stage Imaging Methods Imaging Deep Within Tissue Keeping Cells in Focus Handling the Data Results Conclusion Future Directions 375 376 376 377 o77 Alexander Egner and Stefan W. Hell 377 378 Theory Results of Theoretical Calculations Experiments Other Considerations Dry Objectives Refractive Index, Wavelength, and Temperature Spherical Aberration Correction Conclusion 381 Practical Strategies to Reduce Refractive Index 382 382 ™ CHAPTER ЦІ 382 TOO o„2 .„ ono ono ono ono Biréfringent ono Changes in Emission Spectra Depending on on¿ Microspectroscopy oo7 Light-Specimen Interaction (Fluorescence oo7 Emission) oog -ion The Effect of Fixation on the Optical Properties ZZ of Plants Living Plant Cells од, Stem and Root oc. Microspores од. Storage Structures Mineral Deposits OQ2 Primary and Secondary Cell Walls Fungi ono 393 CHAPTER 393 394 395 ода 395 396 396 398 Contents xvii Signal Level in Confocal Signal-to-Noise Ratio Microscopes QE, N1, N2 and Detectability. 446 Multi-Photon Fluorescence Microscopy. Designs Sampling Comparative Performance of Fluorescence Microscopes Bleaching-Limited Performance Saturation-Limited Performance Effects of Scanning Speed 3D Summary CHAPTER WIDEFIELD/DECONVOLUTION AND CONFOCAL MICROSCOPY FOR THREE- DIMENSIONAL IMAGING Peter J. Shaw Introduction The Point Spread Function: Imaging as a Convolution Limits to Linearity and Shift Deconvolution Practical Differences Temporal Resolution Combination of Charged-Coupled Device and Confocal Imaging Integration of Fluorescence Intensity Resolution, Sensitivity, and Noise Fluorescence Excitation Fluorescent Light Detection Gain Register Charge-Coupled Devices Out-of-Focus Light Model Specimens The Best Solution: Deconvolving Confocal Data Practical Comparisons Conclusion Summary CHAPTER Timothy J. Holmes, David Biggs, and Introduction Purposes of Deconvolution Advantages and Limitations Principles Data Collection Model Maximum Likelihood Estimation Algorithms Different Approaches 3D. 475 2D Image Filtering Data Corrections Light Source and Optics Alignment Newest Developments Subpixel Polarized Light Live Imaging More Examples Blind Deconvolution and Spherical Aberration Widefield Fluorescence Simulation Spinning-Disk Confocal Two Photon Speed Future Directions Summary of Main Points CHAPTER DECONVOLUTION Mark B. Introduction Background Image Formation Forwards: Convolution and the Imaging System Properties of the Point Spread Function Quantifying the Point Spread Function The Missing Cone Problem Noise Deconvolution Algorithms Nearest-Neighbor Deconvolution Wiener Filtering Nonlinear Constrained Iterative Deconvolution Algorithms Comparison of Methods CHAPTER OPTICAL MICROSCOPY Peter Delaney and Martin Harris Introduction Key Fiber Technologies Relevant to Scanning Microscopy Glass Made from Gas and Its Transmission Properties Step Index and Gradient Index Optical Fibers Modes in Optical Fibers Evanescent Wave and Polarization Effects in Optical Fibers Polarization-Maintaining Fibers Fused Biconical Taper Couplers: Fiber-Optic Beam-Splitters Microstructure Fiber Image Transfer Bundles Key Functions of Fibers in Optical Microscopes Optical Fiber for Delivering Light Optical Fiber as a Detection Aperture Same Fiber for Both Source and Confocal Detection Fiber Delivery for Nonlinear Microscopy with Femtosecond Lasers Large Core Fibers as Source or Detection Apertures Benchtop Scanning Microscopes Exploiting Fiber Components Miniaturized Scanning Confocal Microscope Imaging Heads Miniature Confocal Imaging Heads Based on Coherent Imaging Bundles xviii Contents Resolution Bundle Imagers for In Vivo Studies in Animals Scan Heads Based on Single Fibers with Miniature Scanning Mechanisms Vibrating the Fiber Tip Vibrating the Lens and Fiber Scanning with Micromirrors Scanning Fiber Confocal Microscopes for In Vivo Imaging in Animals Implementations for Clinical Endomicroscopy Summary CHAPTER SCANNING MICROSCOPY H.C. Gerritsen, and A. V. Agronskaia Introduction Fluorescence, Lifetime, and Quantum Efficiency Fluorescence Lifetime Spectroscopy Fluorescence Lifetime Imaging Applications Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Methods Introduction Lifetime Sensing in the Frequency Domain Fluorescence Lifetime Sensing in the Time Domain Comparison of Confocal Fluorescence Lifetime Imaging Methods Applications Multi-Labeling and Segmentation Ion-Concentration Determination Probes for Fluorescence Lifetime Microscopy Summary CHAPTER EXCITATION IN LASER-SCANNING MICROSCOPY Winfried Denk, Introduction Physical Principles of Multi-Photon Excitation and Their Implications for Image Formation Physics of Multi-Photon Excitation Optical Pulse Length Excitation Localization Detection Wavelengths Resolution Photodamage: Instrumentation Lasers and the Choice of Excitation Wavelengths Detection Optical Aberrations Pulse Spreading Due to Group Delay Dispersion Control of Laser Power Resonance and Non-Mechanical Scanning Chromophores (Fluorophores and Caged Compounds) Two-Photon Absorption Cross-Sections Caged Compounds Cell Viability During Imaging Applications Calcium Imaging Uncaging and Photobleaching Autofluorescence Developmental Biology In Vivo (Intact Animal) Imaging Outlook CHAPTER MICROSCOPY Jörg Introduction Background Determination of the Optimum Degree of Parallelization Experimental Realization A Multi-Focal Multi-Photon Microscopy Setup Using a Nipkow-Type Microlens Array Resolution Time Multiplexing as a Solution to Crosstalk Alternative Realizations Advanced Variants of Multi-Focal Multi-Photon Microscopy Space Multiplexing Fluorescence Lifetime Imaging Second Harmonic Generation Multi-Focal Multi-Photon Microscopy Multi-Focal Multi-Photon Microscopy-4Pi Microscopy Imaging Applications Limitations Current Developments Summary CHAPTER Jörg Introduction Theoretical Background The Point Spread Function The z-Response and the Axial Resolution The Optical Transfer Function Multi-Focal Multi-Photon Microscopy-^Pi Microscopy Space Live Mammalian Cell 4Pi Imaging Type Resolution Type Summary and Outlook Contents xix CHAPTER FOCUSED DEPLETION AND OTHER REVERSIBLE OPTICAL FLUORESCENCE TRANSITIONS MICROSCOPY CONCEPTS Stefan W. Stefan The Resolution Issue Breaking the Diffraction Barrier: The Concept of Reversible Transitions Different Approaches of Reversible Fluorescence Transitions Microscopy Stimulated Emission Depletion Microscopy Challenges and Outlook CHAPTER AND HARD COPY Guy Cox Introduction Mass Storage Data Compression Removable Storage Media Random-Access Devices Solid State Devices Display Monitors Liquid Crystal Displays Data Projectors Hard Copy Photographic Systems Digital Printers Conclusion Summary Bulk Storage Display Hard Copy CHAPTER RAMAN SCATTERING MICROSCOPY X. Sunney Xie, Ji-Xin Cheng, and Eric Potma Introduction Unique Features of Coherent Anti-Stokes Raman Scattering Under the Tight-Focusing Condition Forward and Backward Detected Coherent Anti-Stokes Raman Scattering Optimal Laser Sources for Coherent Anti-Stokes Raman Scattering Microscopy Suppression of the Non-Resonant Background Use of Picosecond Instead of Femtosecond Pulses Epi-Detection Polarization-Sensitive Detection Time-Resolved Coherent Anti-Stokes Raman Scattering Detection Phase Control of Excitation Pulses Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy Coherent Anti-Stokes Raman Scattering Correlation Spectroscopy Coherent Anti-Stokes Raman Scattering Microscopy Imaging of Biological Samples Conclusions and Perspectives CHAPTER THREE-DIMENSIONAL IMAGING J. Michael Tyszka, Seth W. Ruffins, Jamey P. Michael J. Introduction Surface Imaging Microscopy and Episcopic Fluorescence Image Capture Optical Coherence Tomography Optical Projection Tomography Light Sheet Microscopy Optical Setup Micro-Computerized Tomography Imaging Operating Principle Contrast and Dose Computed Tomography Scanning Systems Magnetic Resonance Microscopy Basic Principles of Nuclear Magnetic Resonance Magnetic Resonance Image Formation Magnetic Resonance Microscopy Hardware Strengths and Limitations of Magnetic Resonance Microscopy Image Contrast in Magnetic Resonance Microscopy Magnetic Resonance Microscopy Applications Future Development of Magnetic Resonance Microscopy Conclusion CHAPTER CONFOCAL MICROSCOPY AND USE OF THE CONFOCAL TEST SPECIMEN Victoria Introduction Getting Started Bleaching Matters Getting a Good Confocal Image Simultaneous Detection of Backscattered Light and Fluorescence New Controls Photon Efficiency Pinhole Size Stray Light Is the Back-Focal Plane Filled? Pinhole Summary Statistical Considerations in Confocal Microscopy The Importance of Pixel Size Measuring Pixel Size Over-Sampling and Under-Sampling Nyquist Reconstruction and Deconvolution Pixel Size Summary xx Contents Using a Test Specimen Why Use a Test Specimen? Description of the Test Specimen Using the Test Specimen The Diatom: A Natural Reasons for Poor Performance Sampling Problems Optical Problems Imaging Depth Singlet-State Saturation Which Optimal Things to Remember About Deconvolution Decision Time Multi-Photon Versus Single-Photon Excitation Widefield Versus Beam Scanning Summary CHAPTER Alan R. Hibbs, Glen MacDonald, and Karl Garsha The Art of Imaging by Confocal Microscopy Balancing Multiple Parameters Monitoring Instrument Performance Illumination Source Scan Raster and Focus Positioning Optical Performance and Objective Lenses Signal Detection Optimizing Multi-Labeling Applications Control Samples Establish the Limits Separation of Fluorescence into Spectral Regions Sequential Channel Collection to Minimize Bleed-Through Spectral Unmixing Colocalization Image Collection for Colocalization Quantifying Colocalization Spatial Deconvolution in Colocalization Studies Discussion CHAPTER MICROSCOPY Jan Huisken, Jim Swoger, Steffen and Ernst H.K. Introduction Combining Light Sheet Illumination and Orthogonal Detection Selective Plane Illumination Microscopy Setup Lateral Resolution Light Sheet Thickness and Axial Resolution Applications Processing Selective Plane Illumination Microscopy Images/Multi-View Reconstruction Summary CHAPTER MULTI-PHOTON MICROSCOPY Karsten König Introduction Photochemical Damage in Multi-Photon Microscopes 680 682 Absorbers and Targets in Biological Specimens Laser Exposure Parameters Evidence for Near Infrared-Induced Reactive Oxygen Species Formation Evidence for Near Infrared-Induced Breaks Photodynamic-Induced Effects Photothermal Damage by Optical Breakdown Modifications of Influence of Reproductive Behavior Nanosurgery Conclusion CHAPTER Alberto Paola Ramoino, and Introduction Photobleaching Photobleaching Reducing Photobleaching Photobleaching of Single Molecules Photobleaching and Photocycling of Single Fluorescent Proteins Bleaching and Autofluorescence Other Fluorescent Proteins Conclusion CHAPTER GENERATION) OPTICAL MICROSCOPY Ping-Chin Cheng and C.K. Sun Introduction Harmonic Generation Second Harmonic Generation Third Harmonic Generation Multi-Photon Absorption and Fluorescence Light Sources and Detectors for Second Harmonic Generation and Third Harmonic Generation Imaging Nonlinear Optical Microscopy Setup Optically Active Biological Structures Optically Active Structures in Plants Optically Active Structures in Animal Tissues Polarization Dependence of Second Harmonic Generation Summary CHAPTER Ayumu Tashiro, Gloster Aaron, Dmitriy Aronov, Rosa Cossart, Daniella Dumitriu, Vivian Fenstermaker, Jesse Goldberg, Farid Hamzei-Sichani, Yuji Ikegaya, Sila Volodymyr Nikolenko, Carlos Portera-Cailliau, and Rafael Yuste Introduction 722 Contents xxi Making Brain Slices Acute Slices Cultured Slices Labeling Cells Biolistic Transfection Genetic Manipulation with Dominant-Negative and Constitutively Active Mutants Diolistics and Caustics Dye Injection with Whole-Cell Patch Clamp Slice Loading and "Painting" with Acetoxymethyl Ester Indicators Green Fluorescent Protein Transgenic Mice Imaging Slices Two-Photon Imaging of Slices Slice Chamber Protocol Choice of Objectives Beam Collimation and Pulse Broadening Image Production, Resolution, and z-Sectioning Choice of Indicators for Two-Photon Imaging of Calcium Photodamage Second Harmonic Imaging Silicon-Intensified Target Camera Imaging Morphological Processing and Analysis Biocytin Protocol Anatomy with a Two-Photon/Neurolucida System Correlated Electron Microscopy Morphological Classification of Neurons Using Cluster Analysis Image Processing Compensation for the Drift and the Vibration of the Slices Alignment Based on the Overlap Between Images Alignment Based on the Center of Mass Online Cell Detection of Neurons Image De-Noising Using Wavelets Summary CHAPTER MEASUREMENT Mark B. Cannell and Stephen H. Cody Introduction The Limiting Case Choice of Indicator Introducing the Indicators into Cells Care of Fluorescent Probes Interpretation of Measurements Kinetics Calibration Conclusion CHAPTER IMAGING OF LIVING EMBRYOS JeffHardin Introduction Into the Depths: Embryos Are Thick, Refractile, and Susceptible to Imaging Embryos Often Requires "4D" Imaging The Quest for Better Resolution: Aberration and the Challenge of Imaging Thick Embryos Embryos Are Highly Scattering and Refractile Specimens Imaging Embryos Involves Inherent Trade-Offs Common Themes in Living Embryo Imaging Have System-Specific Solutions Dealing with Depth: Strategies for Imaging Thick Specimens Avoiding the Thickness Dilemma: Going Small Grazing the Surface: Superficial Optical Sections Are Often Sufficient Up from the Deep: Thickness of Specimens Dramatically Multi-Photon Microscopy Can Penetrate More Deeply into Specimens Selective Plane Illumination Can Provide Optical Sectioning in Very Thick Specimens Deconvolution and Other Post-Acquisition Processing Striving for Speed: Strategies for Reducing Specimen Exposure Simple Solutions: Reducing Image Dimensions, Increasing Slice Spacing, and Scan Speed Disk-Scanning Confocal Microscopy Allows High-Speed Acquisition Additional Hardware Improvements Can Increase Acquisition Speed Localizing Label: Strategies for Increasing Effective Contrast in Thick Specimens Addition of Labeled Proteins to Embryos Expressing Green Fluorescent Protein and mRFP Constructs in Embryos Allows Dynamic Analysis of Embryos at Multiple Wavelengths Using Selective Labeling to Reduce the Number of Labeled Structures Bulk Vital Labeling Can Enhance Contrast Seeing in Space: Strategies for 4D Visualization Depicting Embryos in Time and Space: 2D Versus Other Uses for Confocal and Multi-Photon Microscopy in Imaging and Manipulating Embryos Multi-Photon-Based Ablation Fluorescence Resonance Energy Transfer Conclusions: A Bright Future for Living Embryos CHAPTER Nuno Moreno, Susan Bougourd, and Introduction The Ever Present Problem of Single-Photon Confocal Microscopy Staining Plant Tissues Clearing Intact Plant Material 3D 3D Two-Photon Excitation: Are Two Better Than One? Improved Signal-to-Noise Ratio and Dynamic Range xxii Contents Imaging Thick/Opaque Specimens Fading, Vital Imaging, and Cell Viability Two-Photon Imaging of Plant Cells and Organelles Two-Photon Excitation Imaging of Green Fluorescent Protein Dynamic Imaging Deconvolution Conclusion CHAPTER RESONANCE ENERGY TRANSFER OR MOLECULAR NANOBIOSCOPY OF LIVING CELLS Irina Majoul, Introduction How to Make a Good Science Beauty, Functionality, Cell Cycle, and Living-Cell Imaging Fluorescence Resonance Energy Transfer Theory Fluorescent Proteins and Fluorescence Resonance Energy Transfer Qualitative Analysis Preparation Nanobioscopy of Protein-Protein Interactions with Fluorescence Resonance Energy Transfer Methods of Fluorescence Resonance Energy Transfer Measurement Sensitized Emission of Acceptor Donor Fluorescence Acceptor Bleach Fluorescent Proteins as Fluorescence Resonance Energy Transfer Pairs Cyan Fluorescent Protein and Yellow Fluorescent Protein Resonance Energy Transfer Pair Cyan Fluorescent Protein or Green Fluorescent Protein Forms a Fluorescence Resonance Energy Transfer Pair with mRFPl Fluorescence Resonance Energy Transfer-Based Sensors Fluorescence Resonance Energy Transfer and Other Complementary Methods Fluorescence Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscope Fluorescence Recovery After Photobleaching and Fluorescence Loss in Photobleaching Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Quantum Dots and Fluorescence Resonance Energy Transfer Cloning and Expression of Fluorescent Constructs for Fluorescence Resonance Energy Transfer Cloning of Fluorescent Chimeras Functional Activity of Expressed Constructs Expression and Over-Expression Methods for Introducing Chromophores into Living Cells Electroporation Transfection Reagents Microinjection Future Perspectives: Complexity, and In Vivo Molecular Imaging In Vivo Molecular Imaging CHAPTER IMAGING AND HIGH-CONTENT SCREENING FOR CYTOMICS Maria and Andres Kriete Introduction Platforms Used for Automated Confocal Imaging Types of Assays 3D Data Management and Image Informatics Conclusion CHAPTER SUBCELLULAR LOCATION PATTERNS FROM THREE DIMENSIONAL CONFOCAL MICROSCOPY Ting Zhao and Robert F. Murphy Introduction Protein Subcellular Location Overview of 2D High-Resolution 3DHeLa 3D3T3 Image Acquisition Considerations When Using Automated Analysis Image Processing and Analysis Segmentation of Multi-Cell Images and Preprocessing 3D Automated Classification of Location Patterns Classification of 3DHeLa Downsampled Images with Different Gray Scales Clustering of Location Patterns: Location Proteomics Exclusion of Outliers Determination of Optimal Clustering Statistical Comparison of Location Patterns Image Database Systems Future Directions CHAPTER SOFTWARE Felix Margadant Introduction Testing "Static" Image Performance Brightness Resolution: Changing the Display Size of Your Images Compression Motion Pictures Coding Limitations Contents xxiii Up-Sampling or Frame Rate Matching Motion Picture Artifacts The MPEG Formats MPEG Display Formats Very High Resolutions Movie Compression and Entropy Performance Benchmark Storing Your Presentation for Remote Use Taking Your Presentation on the Road: Digital Rights Management and Overlaying CHAPTER RESOLUTION IS NOT ENOUGH: CORRELATIONAL LIGHT MICROSCOPY AND ELECTRON MICROSCOPY Paul Sims, Ralph Victoria Introduction Early Correlative Microscopy Early 4D Microscopy Correlative Light Microscope/Electron Microscope Today Light Microscope and Electron Microscope Have Different Requirements Finding the Same Cell Structure in Two Different Types of Microscope: Light Microscope/Scanning Electron Microscope Finding the Same Cell Structure in Two Different Types of Microscope: Light Microscope/Transmission Electron Microscope Cryo-Immobilization Followed by Post-Embedding Confocal Laser Scanning Microscopy on Thin Sections Tiled Montage Transmission Electron Microscope Images Aid Correlation Conclusion Metadata Structure Digital Rights Management Future Prospects CHAPTER BIOFILMS — SPATIOTEMPORAL R.J. Palmer, Jr., Janus A.J. Haagensen, Thomas R. Claus Sternberg Introduction Sample Presentation Flowcells and Other Water-Immersible Lenses Upright Versus Inverted Microscopes Setup of a Flow Chamber System Setup Example Making Bacteria Fluorescent Fluorescent Proteins Stains Nucleic Acid Stains Live/Dead Stain Fluorescence In Situ Hybridization General Procedure for Embedding of Flowcell-Grown Biofilms Antibodies Preparation of Labeled Primary Antibodies Imaging Bacteria Without Fluorescence Imaging Extracellular Polymeric Substances in Biofilms Application of Two-Photon Laser-Scanning Microscopy for Limitations of Confocal Laser Scanning Microscopy and Two-Photon Laser-Scanning Microscopy in Biofilm Temporal Experiments Time-Lapse Confocal Imaging Summary and Future Directions CHAPTER THREE-DIMENSIONAL MICROSCOPICAL IMAGES IN BIOLOGY Steffen Introduction Data and Metadata Management in Microscopes Recent Developments Image Information Management The Aims of Modern Microscope System Design Instrument Database Model System Requirements Image Database Model Selected Projects Bioimage. 865 Biomedicai Scientific Image DataBase Other Projects Criteria and Requirements for Microscopy Databases User Interface Query by Content CHAPTER MICROSCOPY Robert H. Webb A. Book and Review Articles B. Historical Interest С D. Technical E. General F. Adaptive Optics G. Differential H. Display I. Fiber-Optic Confocal Microscopes J. Index Mismatch K. Multiplex L. Nonlinear M. Polarization N. O. Point Spread Function P. Pupil Engineering Q. Thickness R. Turbidity S. Variants on the Main Theme xxiv APPENDIX TWO-PHOTON MICROSCOPY Mark B. and Christian Soeller Introduction Laser Safety Laser Alignment Testing Alignment and System Performance Laser Settings and Operation Monitoring Laser Performance Power Levels and Trouble-Shooting Choice of Pulse Length Controlling Laser Power Am I Seeing Two-Photon Excited Fluorescence or Stray Light and Non-Descanned Detection Laser Power Adjustment for Imaging at Depth Simultaneous Imaging of Multiple Labels Minimize Exposure During Orientation and Parameter Setting Ultraviolet-Excited Fluorochromes APPENDIX CURRENT COMMERCIAL CONFOCAL LIGHT MICROSCOPES USED IN BIOLOGY James B. Pawley Introduction BD-CARVII LaVision-BioTec TriM-Scope Leica Nikon Clsi Olympus Fluoview 1000-DSU Visitech VT Infmity-VT-eye Yokogawa Zeiss LSM-5-LIVE Fast Slit Scanner-LSM META-FCS APPENDIX TO KNOW ABOUT CHARGE-COUPLED DEVICES James B. Pawley Introduction Part Charge Coupling Readout Methods What Could Go Wrong? Quantum Efficiency Edge Effects Charge Loss Leakage or "Dark Charge" Blooming Incomplete Charge Transfer Charge Amplifiers What Is a Charge Amplifier? FET Noise Sources in the Charge-Coupled Device Fixed Pattern Noise Noise from the Charge Amplifier Where Is Zero? A New Idea: The Gain Register Amplifier!! Of Course, There Is One Snag! Part II: Evaluating a Charge-Coupled Device A. Important Charge-Coupled Device Specs for Live-Cell Stuff! B. Things That Are (Almost!) Irrelevant When Choosing a Charge-Coupled Device for Live-Cell Microscopy C. A Test You Can Do Yourself!!! D. Intensified Charge-Coupled Devices Index
adam_txt	Contents Preface to the Third Edition Preface to the Second Edition Contributors CHAPTER CONFOCAL SCANNED IMAGING IN LIGHT MICROSCOPY Shinya Light Microscopy Lateral Resolution Axial Resolution Depth of Field Confocal Imaging Impact of Video Nipkow Disk Electron-Beam-Scanning Television Impact of Modern Video Lasers and Microscopy Holography Laser Illumination Laser-Illuminated Confocal Microscopes Confocal Laser-Scanning Microscope Two- and Multi-Photon Microscopy Is Laser-Scanning Confocal Microsopy a Cure-Ail? Speed of Image or Data Acquisition Yokogawa Disk-Scanning Confocal System Depth of Field in Phase-Dependent Imaging Other Optical and Mechanical Factors Affecting Confocal Microscopy Lens Aberration Unintentional Beam Deviation Contrast Transfer and Resolution in Confocal Versus Non-Confocal Microscopy Summary CHAPTER CONFOCAL MICROSCOPY James B. Pawley Introduction What Limits? Counting Statistics: The Importance of Source Brightness Specimen Response: Dye Saturation A Typical Problem Practical Photon Efficiency Losses in the Optical System Detection and Measurement Losses Where Have All the Photons Gone? Resolution: How Much Is Enough? Can Resolution Be Too High? Limitations Imposed by Spatial and Temporal Quantization Practical Considerations Relating Resolution to Distortion Conclusion CHAPTER Jens Rietdorf Introduction Regulating the Intensity Wavelength Selective Filtering Devices Selecting the Wavelength of the Illumination and the Detected Light Separating the Light Paths Conventional Filters Interference Filters Dichroic and Polarizing Beam-Splitters Filters and Dispersive Elements for Multi-Channel Detection Mechanical Scanners Galvanometer Scanners General Specifications Acousto-Optical Components Acousto-Optical Deflectors Acousto-Optical Modulators Acousto-Optical Tunable Filters Acousto-Optical Beam-Splitters Electro-Optical Modulators Piezoelectric Scanners Polarizing Elements Removing Excess Light CHAPTER LEVELS: DIGITIZING IMAGE DATA James B. Pawley Contrast Transfer Function, Points, and Pixels Pixels, Images, and the Contrast Transfer Function Digitization and Pixels Digitization of Images How Big Should a Pixel Be? Sampling and Quantum Noise The Nyquist Criterion Estimating the Expected Resolution of an Image The Story So Far Reality Check? Is Over-Sampling Ever Wise? Under-Sampling? Digitizing Trade-Offs xii Contents Nyquist Some Gray Levels, "Noise," and Photodetector Performance Optical Density The Zone System: Quantified Photography Linearity: Do We Need It? Gray Levels in Images Recorded Using Charge-Coupled Devices: The Intensity Spread Function What Counts as Noise? Measuring the Intensity Spread Function Calibrating a Charge-Coupled Device to Measure the ISF "Fixed-Pattern" Noise Gain-Register Charge-Coupled Devices Multiplicative Noise Trade-Offs CHAPTER CONFOCAL MICROSCOPY Enrico Gratton and Martin J. vandeVen Introduction Laser Power Requirements The Basic Laser Principle of Operation Pumping Power Requirements Laser Modes: Longitudinal (Axial) and Transverse Polarization Coherent Properties of Laser Light Phase Randomization: Scrambling the Coherence Properties of Laser Light Measures to Reduce the Coherence Length of Laser Light Heat Removal Other Installation Requirements Attenuation of Laser Beams Stabilization of Intensity, Wavelength, and Beam Position in Lasers Sources of Noise in Lasers Spatial Beam Characteristics Laser Requirements for Biological Confocal Laser Scanning Microscopy-Related Techniques Optical Tweezers Total Internal Reflection Microscopy Confocal Raman Confocal Laser Scanning Microscopy for Chemical Imaging Non-Linear Confocal Microscopy Nanosurgery and Types of Lasers Continuous Wave Lasers Gas Lasers Dye Lasers Solid-State Lasers Thin Disk Lasers Pulsed Lasers Classification of Pulsed Laser Systems Nitrogen Lasers Excimer Lasers Metal Vapor Lasers Dye Lasers Modulated Diode Lasers Diode Pumped Solid State Laser in Pulsed Mode Ultrafast Diode Pumped Solid State Lasers Titanium-Sapphire and Related Ultrafast Lasers White Light Continuum Lasers Ultrafast Fiber Lasers Wavelength Expansion Through Non-Linear Techniques Second and Higher Harmonic Generation: SHG, THG, FHG Label-Free Microscopy Sum or Difference Mixing Optical Parametric Oscillators and Optical Parametric Amplifiers Pulse Length Measurement Maintenance Maintenance of Active Laser Media Maintenance of Pumping Media Maintenance of the Optical Resonator Maintenance of Other System Components Troubleshooting Safety Precautions Beam Stops Curtains Laser Goggles Screens Exposure Effects, Warning Signs, and Interlocks Infrared Paper Conclusion CHAPTER FOR THREE-DIMENSIONAL MICROSCOPY Andreas Nolte, James B. Pawley, and Introduction General Remarks on Choice of Excitation Light Sources Scrambling and Filtering the Light Types of Sources and Their Features Structure Wavelength Stability in Time and Wavelength Radiance Control Measuring What Comes Through the Illumination System The Bare Minimum Types of Confocal Microscopes That Can Use Non-Laser Light Sources Tandem Scanning: Basic Description Single-Sided Disk Scanning: Basic Description Exposure Time and Source Brightness Future Trends CHAPTER CONFOCAL MICROSCOPY H. Ernst Keller Introduction Aberrations of Refractive Systems Defocusing Monochromatic Aberrations Chromatic Aberrations Contents xiii Finite Working Distance Optical Materials Anti-Reflection Coatings Transmission of Microscope Objectives Conclusion CHAPTER IN OPTICAL MICROSCOPY Ping-Chin Cheng Introduction Sources of Contrast Absorption Contrast Scattering and Reflection Contrast Phase Contrast Fluorescence Contrast Contrast Related to Excitation Wavelength Change Negative Contrast Special Concerns in Ultraviolet and Near-Infrared Range Confocal Microscopy Total Internal Reflection Contrast Harmonic Generation Contrast Geometric Contrast z-Contrast in Confocal Microscopy Total Internal Refraction Fluorescence Contrast Fluorescence Resonant Energy Transfer Fluorescence Recovery After Photobleaching (FRAP and FLIP) Structural Contrast Harmonic Generation Contrast Birefringence Contrast Derived Contrast (Synthetic Contrast) Ratiometric Deconvolution Movement Contrast (Subtraction of Previous Image) Spectral Unmixing and Color Reassignment Effects of the Specimen: Spherical Aberration and Optical Heterogeneity Mounting Medium Selection Artificial Contrast Contrast Resulting from Instrument Vibration and Ambient Lighting Contrast Resulting from Interference of Cover Glass Surfaces Background Level and Ghost Images from the Transmission Illuminator Contrast Resulting from Differences in Photobleaching Dynamics Effect of Spectral Leakage and Signal Imbalance Between Different Channels New Contrasts: Fluorescence Lifetime and Coherent Antistokes Raman Spectroscopy Summary CHAPTER SYSTEM OF LASER-SCANNING CONFOCAL MICROSCOPES Ernst H.K. Introduction Design Principles Overview Telecentricity The Scanning System The Back-Focal Planes Practical Requirements Diffraction Limit Geometric Distortion Evaluation of the Illumination and Detection Systems Influence of Optical Elements Errors Evaluation of Optical Arrangements Evaluation of Scanner Arrangements Scanners Attachment to Microscopes Merit Functions Multi-Fluorescence Special Setups Setups for Fluorescence Recovery After Photobleaching Experiments Setups for Fluorescence Resonance Energy Transfer Experiments Setups for the Integration of Optical Tweezers Setups for the Integration of Laser Cutters Setups for the Observation of Living Specimens Miniaturization and Computer Control Thermal Stability Vibration Isolation Conclusions and Future Prospects CHAPTER MICROSCOPY Derek Toomre and James B. Pawley Introduction Background Living Cell Imaging: Probing the Future A Need for Speed and Less Photobleaching Advantages and Limitations of Confocal Laser-Scanning Microscopes Other Imaging and Deconvolution Confocal Disk-Scanning Microscopy Nipkow Disk A Renaissance Confocal Imaging Disadvantages Critical Parameters in Pinhole and Slit Disks Fill Factor and Spacing Interval Lateral Resolution Pinhole/Slit Size Axial Resolution Types of Disk-Scanning General Considerations Disk Scanners for Backscattered Light Imaging CARV, Microscopes The Yokogawa Microlens Approach New Fast Slit Scanner New Detectors Image Intensifies On-Chip Electron Multiplying Charge-Coupled Device xiv Contents Electron Disk Scanners Applications and Examples of Confocal Disk-Scanning Microscopes Comparison with Epi-Fluorescence Imaging Fast 3D/4D Imaging Blazingly Fast Confocal Imaging Future Developments? Summary CHAPTER FUNCTION OF HIGH NUMERICAL APERTURE MICROSCOPE OBJECTIVE LENSES Rimas Introduction Measuring Point Spread Function Fiber-Optic Interferometer Point Spread Function Measurements Chromatic Aberrations Apparatus Axial Shift Pupil Function Phase-Shifting Zernike Polynomial Fit Restoration of a Empty Aperture Miscellanea Temperature Variations Polarization Effects Apodization Conclusion CHAPTER CONFOCAL MICROSCOPY Jonathan Art Introduction The Quantal Nature of Light Interaction of Photons with Materials Thermal Effects Direct Effects Photoconductivity Photovoltaic Photoemissive Comparison of Detectors Noise Internal to Detectors Noise in Internal Detectors Noise in Photoemissive Devices Statistics of Photon Flux and Detectors Representing the Pixel Value Conversion Techniques Assessment of Devices Point Detection Assessment and Optimization Field Detection Assessment and Optimization Detectors Present and Future CHAPTER METHODS Rainer Heintzmann Introduction 265 Experimental Considerations Pattern Generation Computing Optical Sections from Structured-Illumination Data Resolution Improvement by Structured Illumination Nonlinear Structured Illumination Summary CHAPTER MULTI-DIMENSIONAL MICROSCOPY IMAGES N.S. White Introduction Definitions What Is the Microscopist Trying to Achieve? Criteria for Choosing a Visualization System Why Do We Want to Visualize Multi-Dimensional Laser-Scanning Microscopy Data? Data and Dimensional Reduction Objective or Subjective Visualization? Prefiltering Identifying Unknown Structures Highlighting Previously Elucidated Structures Visualization for Multi-Dimensional Measurements What Confocal Laser Scanning Microscopy Images Can the Visualization System Handle? Image Data: How Are Image Values Represented in the Program? What Dimensions Can the Images and Views Have? Standard File Formats for Calibration and Interpretation How Will the System Generate the Reconstructed Views? Assessing the Four Basic Steps in the Generation of Reconstructed Views Loading the Image Subregion Choosing a View: The 5D Image Display Space Mapping the Image Space into the Display Space How Do z-Information? Mapping the Data Values into the Display How Can Intensities Be Used to Retain г Hidden-Object Removal Adding Realism to the View How Can I Make Measurements Using the Reconstructed Views? Conclusion CHAPTER DIMENSIONAL IMAGE ANALYSIS METHODS FOR CONFOCAL MICROSCOPY Badrinath Omar Al-Kofahi, Khalid Al-Kofahi, William Shain, Donald H. Szarowsk, and James Introduction Types of Automated Image Analysis Studies Contents xv Common Types of Biological Image Objects Specimen Preparation and Image Preprocessing Methods Data Collection Guidelines for Image Analysis Purposes Image Preprocessing Methods General Segmentation Methods Applicable to Confocal Data Bottom-Up Segmentation Methods Top-Down Segmentation Methods Hybrid Segmentation Methods Combining Bottom-Up and Top-Down Processing Example Illustrating Blob Segmentation Model-Based Object Merging Example Illustrating Segmentation of Tube-Like Objects Skeletonization Methods Vectorization Methods Example Combining Tube and Blob Segmentation Registration and Montage Synthesis Methods Methods for Quantitative Morphometry Methods for Validating the Segmentation and Making Corrections Analysis of Morphometric Data Discussion, Conclusion, and Future Directions CHAPTER MICROSCOPY: PHOTOPHYSICS AND PHOTOCHEMISTRY Roger Y. Tsien, Lauren Ernst, and Alan Waggoner Introduction Photophysical Problems Related to High Intensity Excitation Singlet State Saturation Triplet State Saturation Contaminating Background Signals What Is the Optimal Intensity? Photodestruction Specimens Dependency on Intensity or Its Time Integral? Strategies for Signal Optimization in the Face of Photobleaching Light Collection Efficiency Spatial Resolution Protective Agents Fluorophore Concentration Choice of Fluorophore Fluorescent Labels for Antibodies, Other Proteins, and Fluorescent Organic Dyes Phycobiliproteins DNA Luminescent Nanocrystals Fluorescent Lanthanide Chelates Fluorescent Indicators for Dynamic Intracellular Parameters Membrane Potentials Ion Concentrations pH Ca2+ Indicators Oxygen Sensor cAMP Indicators Fatty Acid Indicator Genetically Expressed Intracellular Fluorescent Indicators Green Fluorescent Protein Ligand-Binding Modules Ion Indicators Future Developments CHAPTER THE SELECTION AND APPLICATION OF FLUORESCENT PROBES Iain D. Johnson Introduction Selection Criteria for Dyes and Probes Organic Dyes Fluorescent Proteins: Green Fluorescent Protein and Phycobiliproteins Quantum Dots Multi-Photon Excitation Introducing the Probe to the Specimen Loading Methods Target Abundance and Considerations Interactions of Probes and Specimens Localization and Metabolism Perturbation and Cytotoxicity Under the Microscope Photobleaching Phototoxicity Summary CHAPTER SPECIMEN PRESERVATION FOR CONFOCAL FLUORESCENCE MICROSCOPY Robert Introduction Characteristics of Fixatives Glutaraldehyde Formaldehyde Fixation Staining and Mounting Methods Glutaraldehyde Fixation pH Immunofluorescence Staining Mounting the Specimen Critical Evaluation of Light Microscopy Fixation and Mounting Methods Use of the Cell Height to Evaluate the Fixation Method Use of Cell Height to Evaluate Mounting Media Well-Defined Structures Can Be Used to Evaluate Fixation Methods Comparison of In Vivo Labeled Cell Immunolabeled Cell General Notes Labeling Samples with Two or More Probes xvi Contents Triple Preparation of Tissue Specimens Labeling Thick Sections Refractive Index Mismatch Screening Antibodies on Glutaraldehyde-Fixed Specimens Microwave Fixation Conclusion CHAPTER OF LIVING CELLS Michael E. Dailey, Erik Manders, David R. Soil, and Mark Terasaki Introduction Overview of Living-Cell Confocal Imaging Techniques Time-Lapse Fluorescence Imaging Multi-Channel Time-Lapse Fluorescence Imaging Spectral Imaging and Linear Unmixing Fluorescence Recovery After Photobleaching Fluorescence Loss in Photobleaching Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Fluorescence Correlation Spectroscopy Fluorescence Photo-Uncaging/Photoactivation Optical Tweezers/Laser Trapping Physiological Fluorescence Imaging Combining Fluorescence and Other Imaging Modalities General Considerations for Confocal Microscopy of Living Cells Maintenance of Living Cells and Tissue Preparations Fluorescent Probes Minimizing Photodynamic Damage The Online Confocal Community A Convenient Test Specimen Specific Example I: Visualizing Chromatin Dynamics Using Very Low Light Levels Phototoxicity Reduction of Phototoxicity Improving Image Quality in Low-Dose Microscopy Low-Dose Imaging Conclusion Specific Example II: Multi-Dimensional Imaging of Microglial Cell Behaviors in Live Rodent Brain Slices Preparation of Central Nervous System Tissue Slices Fluorescent Staining Maintaining Tissue Health on the Microscope Stage Imaging Methods Imaging Deep Within Tissue Keeping Cells in Focus Handling the Data Results Conclusion Future Directions 375 376 376 377 o77 Alexander Egner and Stefan W. Hell 377 378 Theory Results of Theoretical Calculations Experiments Other Considerations Dry Objectives Refractive Index, Wavelength, and Temperature Spherical Aberration Correction Conclusion 381 Practical Strategies to Reduce Refractive Index 382 382 ™ CHAPTER ЦІ 382 TOO o„2 .„ ono ono ono ono Biréfringent ono Changes in Emission Spectra Depending on on¿ Microspectroscopy oo7 Light-Specimen Interaction (Fluorescence oo7 Emission) oog -ion The Effect of Fixation on the Optical Properties ZZ of Plants Living Plant Cells од, Stem and Root oc. Microspores од. Storage Structures Mineral Deposits OQ2 Primary and Secondary Cell Walls Fungi ono 393 CHAPTER 393 394 395 ода 395 396 396 398 Contents xvii Signal Level in Confocal Signal-to-Noise Ratio Microscopes QE, N1, N2 and Detectability. 446 Multi-Photon Fluorescence Microscopy. Designs Sampling Comparative Performance of Fluorescence Microscopes Bleaching-Limited Performance Saturation-Limited Performance Effects of Scanning Speed 3D Summary CHAPTER WIDEFIELD/DECONVOLUTION AND CONFOCAL MICROSCOPY FOR THREE- DIMENSIONAL IMAGING Peter J. Shaw Introduction The Point Spread Function: Imaging as a Convolution Limits to Linearity and Shift Deconvolution Practical Differences Temporal Resolution Combination of Charged-Coupled Device and Confocal Imaging Integration of Fluorescence Intensity Resolution, Sensitivity, and Noise Fluorescence Excitation Fluorescent Light Detection Gain Register Charge-Coupled Devices Out-of-Focus Light Model Specimens The Best Solution: Deconvolving Confocal Data Practical Comparisons Conclusion Summary CHAPTER Timothy J. Holmes, David Biggs, and Introduction Purposes of Deconvolution Advantages and Limitations Principles Data Collection Model Maximum Likelihood Estimation Algorithms Different Approaches 3D. 475 2D Image Filtering Data Corrections Light Source and Optics Alignment Newest Developments Subpixel Polarized Light Live Imaging More Examples Blind Deconvolution and Spherical Aberration Widefield Fluorescence Simulation Spinning-Disk Confocal Two Photon Speed Future Directions Summary of Main Points CHAPTER DECONVOLUTION Mark B. Introduction Background Image Formation Forwards: Convolution and the Imaging System Properties of the Point Spread Function Quantifying the Point Spread Function The Missing Cone Problem Noise Deconvolution Algorithms Nearest-Neighbor Deconvolution Wiener Filtering Nonlinear Constrained Iterative Deconvolution Algorithms Comparison of Methods CHAPTER OPTICAL MICROSCOPY Peter Delaney and Martin Harris Introduction Key Fiber Technologies Relevant to Scanning Microscopy Glass Made from Gas and Its Transmission Properties Step Index and Gradient Index Optical Fibers Modes in Optical Fibers Evanescent Wave and Polarization Effects in Optical Fibers Polarization-Maintaining Fibers Fused Biconical Taper Couplers: Fiber-Optic Beam-Splitters Microstructure Fiber Image Transfer Bundles Key Functions of Fibers in Optical Microscopes Optical Fiber for Delivering Light Optical Fiber as a Detection Aperture Same Fiber for Both Source and Confocal Detection Fiber Delivery for Nonlinear Microscopy with Femtosecond Lasers Large Core Fibers as Source or Detection Apertures Benchtop Scanning Microscopes Exploiting Fiber Components Miniaturized Scanning Confocal Microscope Imaging Heads Miniature Confocal Imaging Heads Based on Coherent Imaging Bundles xviii Contents Resolution Bundle Imagers for In Vivo Studies in Animals Scan Heads Based on Single Fibers with Miniature Scanning Mechanisms Vibrating the Fiber Tip Vibrating the Lens and Fiber Scanning with Micromirrors Scanning Fiber Confocal Microscopes for In Vivo Imaging in Animals Implementations for Clinical Endomicroscopy Summary CHAPTER SCANNING MICROSCOPY H.C. Gerritsen, and A. V. Agronskaia Introduction Fluorescence, Lifetime, and Quantum Efficiency Fluorescence Lifetime Spectroscopy Fluorescence Lifetime Imaging Applications Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Methods Introduction Lifetime Sensing in the Frequency Domain Fluorescence Lifetime Sensing in the Time Domain Comparison of Confocal Fluorescence Lifetime Imaging Methods Applications Multi-Labeling and Segmentation Ion-Concentration Determination Probes for Fluorescence Lifetime Microscopy Summary CHAPTER EXCITATION IN LASER-SCANNING MICROSCOPY Winfried Denk, Introduction Physical Principles of Multi-Photon Excitation and Their Implications for Image Formation Physics of Multi-Photon Excitation Optical Pulse Length Excitation Localization Detection Wavelengths Resolution Photodamage: Instrumentation Lasers and the Choice of Excitation Wavelengths Detection Optical Aberrations Pulse Spreading Due to Group Delay Dispersion Control of Laser Power Resonance and Non-Mechanical Scanning Chromophores (Fluorophores and Caged Compounds) Two-Photon Absorption Cross-Sections Caged Compounds Cell Viability During Imaging Applications Calcium Imaging Uncaging and Photobleaching Autofluorescence Developmental Biology In Vivo (Intact Animal) Imaging Outlook CHAPTER MICROSCOPY Jörg Introduction Background Determination of the Optimum Degree of Parallelization Experimental Realization A Multi-Focal Multi-Photon Microscopy Setup Using a Nipkow-Type Microlens Array Resolution Time Multiplexing as a Solution to Crosstalk Alternative Realizations Advanced Variants of Multi-Focal Multi-Photon Microscopy Space Multiplexing Fluorescence Lifetime Imaging Second Harmonic Generation Multi-Focal Multi-Photon Microscopy Multi-Focal Multi-Photon Microscopy-4Pi Microscopy Imaging Applications Limitations Current Developments Summary CHAPTER Jörg Introduction Theoretical Background The Point Spread Function The z-Response and the Axial Resolution The Optical Transfer Function Multi-Focal Multi-Photon Microscopy-^Pi Microscopy Space Live Mammalian Cell 4Pi Imaging Type Resolution Type Summary and Outlook Contents xix CHAPTER FOCUSED DEPLETION AND OTHER REVERSIBLE OPTICAL FLUORESCENCE TRANSITIONS MICROSCOPY CONCEPTS Stefan W. Stefan The Resolution Issue Breaking the Diffraction Barrier: The Concept of Reversible Transitions Different Approaches of Reversible Fluorescence Transitions Microscopy Stimulated Emission Depletion Microscopy Challenges and Outlook CHAPTER AND HARD COPY Guy Cox Introduction Mass Storage Data Compression Removable Storage Media Random-Access Devices Solid State Devices Display Monitors Liquid Crystal Displays Data Projectors Hard Copy Photographic Systems Digital Printers Conclusion Summary Bulk Storage Display Hard Copy CHAPTER RAMAN SCATTERING MICROSCOPY X. Sunney Xie, Ji-Xin Cheng, and Eric Potma Introduction Unique Features of Coherent Anti-Stokes Raman Scattering Under the Tight-Focusing Condition Forward and Backward Detected Coherent Anti-Stokes Raman Scattering Optimal Laser Sources for Coherent Anti-Stokes Raman Scattering Microscopy Suppression of the Non-Resonant Background Use of Picosecond Instead of Femtosecond Pulses Epi-Detection Polarization-Sensitive Detection Time-Resolved Coherent Anti-Stokes Raman Scattering Detection Phase Control of Excitation Pulses Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy Coherent Anti-Stokes Raman Scattering Correlation Spectroscopy Coherent Anti-Stokes Raman Scattering Microscopy Imaging of Biological Samples Conclusions and Perspectives CHAPTER THREE-DIMENSIONAL IMAGING J. Michael Tyszka, Seth W. Ruffins, Jamey P. Michael J. Introduction Surface Imaging Microscopy and Episcopic Fluorescence Image Capture Optical Coherence Tomography Optical Projection Tomography Light Sheet Microscopy Optical Setup Micro-Computerized Tomography Imaging Operating Principle Contrast and Dose Computed Tomography Scanning Systems Magnetic Resonance Microscopy Basic Principles of Nuclear Magnetic Resonance Magnetic Resonance Image Formation Magnetic Resonance Microscopy Hardware Strengths and Limitations of Magnetic Resonance Microscopy Image Contrast in Magnetic Resonance Microscopy Magnetic Resonance Microscopy Applications Future Development of Magnetic Resonance Microscopy Conclusion CHAPTER CONFOCAL MICROSCOPY AND USE OF THE CONFOCAL TEST SPECIMEN Victoria Introduction Getting Started Bleaching Matters Getting a Good Confocal Image Simultaneous Detection of Backscattered Light and Fluorescence New Controls Photon Efficiency Pinhole Size Stray Light Is the Back-Focal Plane Filled? Pinhole Summary Statistical Considerations in Confocal Microscopy The Importance of Pixel Size Measuring Pixel Size Over-Sampling and Under-Sampling Nyquist Reconstruction and Deconvolution Pixel Size Summary xx Contents Using a Test Specimen Why Use a Test Specimen? Description of the Test Specimen Using the Test Specimen The Diatom: A Natural Reasons for Poor Performance Sampling Problems Optical Problems Imaging Depth Singlet-State Saturation Which Optimal Things to Remember About Deconvolution Decision Time Multi-Photon Versus Single-Photon Excitation Widefield Versus Beam Scanning Summary CHAPTER Alan R. Hibbs, Glen MacDonald, and Karl Garsha The Art of Imaging by Confocal Microscopy Balancing Multiple Parameters Monitoring Instrument Performance Illumination Source Scan Raster and Focus Positioning Optical Performance and Objective Lenses Signal Detection Optimizing Multi-Labeling Applications Control Samples Establish the Limits Separation of Fluorescence into Spectral Regions Sequential Channel Collection to Minimize Bleed-Through Spectral Unmixing Colocalization Image Collection for Colocalization Quantifying Colocalization Spatial Deconvolution in Colocalization Studies Discussion CHAPTER MICROSCOPY Jan Huisken, Jim Swoger, Steffen and Ernst H.K. Introduction Combining Light Sheet Illumination and Orthogonal Detection Selective Plane Illumination Microscopy Setup Lateral Resolution Light Sheet Thickness and Axial Resolution Applications Processing Selective Plane Illumination Microscopy Images/Multi-View Reconstruction Summary CHAPTER MULTI-PHOTON MICROSCOPY Karsten König Introduction Photochemical Damage in Multi-Photon Microscopes 680 682 Absorbers and Targets in Biological Specimens Laser Exposure Parameters Evidence for Near Infrared-Induced Reactive Oxygen Species Formation Evidence for Near Infrared-Induced Breaks Photodynamic-Induced Effects Photothermal Damage by Optical Breakdown Modifications of Influence of Reproductive Behavior Nanosurgery Conclusion CHAPTER Alberto Paola Ramoino, and Introduction Photobleaching Photobleaching Reducing Photobleaching Photobleaching of Single Molecules Photobleaching and Photocycling of Single Fluorescent Proteins Bleaching and Autofluorescence Other Fluorescent Proteins Conclusion CHAPTER GENERATION) OPTICAL MICROSCOPY Ping-Chin Cheng and C.K. Sun Introduction Harmonic Generation Second Harmonic Generation Third Harmonic Generation Multi-Photon Absorption and Fluorescence Light Sources and Detectors for Second Harmonic Generation and Third Harmonic Generation Imaging Nonlinear Optical Microscopy Setup Optically Active Biological Structures Optically Active Structures in Plants Optically Active Structures in Animal Tissues Polarization Dependence of Second Harmonic Generation Summary CHAPTER Ayumu Tashiro, Gloster Aaron, Dmitriy Aronov, Rosa Cossart, Daniella Dumitriu, Vivian Fenstermaker, Jesse Goldberg, Farid Hamzei-Sichani, Yuji Ikegaya, Sila Volodymyr Nikolenko, Carlos Portera-Cailliau, and Rafael Yuste Introduction 722 Contents xxi Making Brain Slices Acute Slices Cultured Slices Labeling Cells Biolistic Transfection Genetic Manipulation with Dominant-Negative and Constitutively Active Mutants Diolistics and Caustics Dye Injection with Whole-Cell Patch Clamp Slice Loading and "Painting" with Acetoxymethyl Ester Indicators Green Fluorescent Protein Transgenic Mice Imaging Slices Two-Photon Imaging of Slices Slice Chamber Protocol Choice of Objectives Beam Collimation and Pulse Broadening Image Production, Resolution, and z-Sectioning Choice of Indicators for Two-Photon Imaging of Calcium Photodamage Second Harmonic Imaging Silicon-Intensified Target Camera Imaging Morphological Processing and Analysis Biocytin Protocol Anatomy with a Two-Photon/Neurolucida System Correlated Electron Microscopy Morphological Classification of Neurons Using Cluster Analysis Image Processing Compensation for the Drift and the Vibration of the Slices Alignment Based on the Overlap Between Images Alignment Based on the Center of Mass Online Cell Detection of Neurons Image De-Noising Using Wavelets Summary CHAPTER MEASUREMENT Mark B. Cannell and Stephen H. Cody Introduction The Limiting Case Choice of Indicator Introducing the Indicators into Cells Care of Fluorescent Probes Interpretation of Measurements Kinetics Calibration Conclusion CHAPTER IMAGING OF LIVING EMBRYOS JeffHardin Introduction Into the Depths: Embryos Are Thick, Refractile, and Susceptible to Imaging Embryos Often Requires "4D" Imaging The Quest for Better Resolution: Aberration and the Challenge of Imaging Thick Embryos Embryos Are Highly Scattering and Refractile Specimens Imaging Embryos Involves Inherent Trade-Offs Common Themes in Living Embryo Imaging Have System-Specific Solutions Dealing with Depth: Strategies for Imaging Thick Specimens Avoiding the Thickness Dilemma: Going Small Grazing the Surface: Superficial Optical Sections Are Often Sufficient Up from the Deep: Thickness of Specimens Dramatically Multi-Photon Microscopy Can Penetrate More Deeply into Specimens Selective Plane Illumination Can Provide Optical Sectioning in Very Thick Specimens Deconvolution and Other Post-Acquisition Processing Striving for Speed: Strategies for Reducing Specimen Exposure Simple Solutions: Reducing Image Dimensions, Increasing Slice Spacing, and Scan Speed Disk-Scanning Confocal Microscopy Allows High-Speed Acquisition Additional Hardware Improvements Can Increase Acquisition Speed Localizing Label: Strategies for Increasing Effective Contrast in Thick Specimens Addition of Labeled Proteins to Embryos Expressing Green Fluorescent Protein and mRFP Constructs in Embryos Allows Dynamic Analysis of Embryos at Multiple Wavelengths Using Selective Labeling to Reduce the Number of Labeled Structures Bulk Vital Labeling Can Enhance Contrast Seeing in Space: Strategies for 4D Visualization Depicting Embryos in Time and Space: 2D Versus Other Uses for Confocal and Multi-Photon Microscopy in Imaging and Manipulating Embryos Multi-Photon-Based Ablation Fluorescence Resonance Energy Transfer Conclusions: A Bright Future for Living Embryos CHAPTER Nuno Moreno, Susan Bougourd, and Introduction The Ever Present Problem of Single-Photon Confocal Microscopy Staining Plant Tissues Clearing Intact Plant Material 3D 3D Two-Photon Excitation: Are Two Better Than One? Improved Signal-to-Noise Ratio and Dynamic Range xxii Contents Imaging Thick/Opaque Specimens Fading, Vital Imaging, and Cell Viability Two-Photon Imaging of Plant Cells and Organelles Two-Photon Excitation Imaging of Green Fluorescent Protein Dynamic Imaging Deconvolution Conclusion CHAPTER RESONANCE ENERGY TRANSFER OR MOLECULAR NANOBIOSCOPY OF LIVING CELLS Irina Majoul, Introduction How to Make a Good Science Beauty, Functionality, Cell Cycle, and Living-Cell Imaging Fluorescence Resonance Energy Transfer Theory Fluorescent Proteins and Fluorescence Resonance Energy Transfer Qualitative Analysis Preparation Nanobioscopy of Protein-Protein Interactions with Fluorescence Resonance Energy Transfer Methods of Fluorescence Resonance Energy Transfer Measurement Sensitized Emission of Acceptor Donor Fluorescence Acceptor Bleach Fluorescent Proteins as Fluorescence Resonance Energy Transfer Pairs Cyan Fluorescent Protein and Yellow Fluorescent Protein Resonance Energy Transfer Pair Cyan Fluorescent Protein or Green Fluorescent Protein Forms a Fluorescence Resonance Energy Transfer Pair with mRFPl Fluorescence Resonance Energy Transfer-Based Sensors Fluorescence Resonance Energy Transfer and Other Complementary Methods Fluorescence Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscope Fluorescence Recovery After Photobleaching and Fluorescence Loss in Photobleaching Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Quantum Dots and Fluorescence Resonance Energy Transfer Cloning and Expression of Fluorescent Constructs for Fluorescence Resonance Energy Transfer Cloning of Fluorescent Chimeras Functional Activity of Expressed Constructs Expression and Over-Expression Methods for Introducing Chromophores into Living Cells Electroporation Transfection Reagents Microinjection Future Perspectives: Complexity, and In Vivo Molecular Imaging In Vivo Molecular Imaging CHAPTER IMAGING AND HIGH-CONTENT SCREENING FOR CYTOMICS Maria and Andres Kriete Introduction Platforms Used for Automated Confocal Imaging Types of Assays 3D Data Management and Image Informatics Conclusion CHAPTER SUBCELLULAR LOCATION PATTERNS FROM THREE DIMENSIONAL CONFOCAL MICROSCOPY Ting Zhao and Robert F. Murphy Introduction Protein Subcellular Location Overview of 2D High-Resolution 3DHeLa 3D3T3 Image Acquisition Considerations When Using Automated Analysis Image Processing and Analysis Segmentation of Multi-Cell Images and Preprocessing 3D Automated Classification of Location Patterns Classification of 3DHeLa Downsampled Images with Different Gray Scales Clustering of Location Patterns: Location Proteomics Exclusion of Outliers Determination of Optimal Clustering Statistical Comparison of Location Patterns Image Database Systems Future Directions CHAPTER SOFTWARE Felix Margadant Introduction Testing "Static" Image Performance Brightness Resolution: Changing the Display Size of Your Images Compression Motion Pictures Coding Limitations Contents xxiii Up-Sampling or Frame Rate Matching Motion Picture Artifacts The MPEG Formats MPEG Display Formats Very High Resolutions Movie Compression and Entropy Performance Benchmark Storing Your Presentation for Remote Use Taking Your Presentation on the Road: Digital Rights Management and Overlaying CHAPTER RESOLUTION IS NOT ENOUGH: CORRELATIONAL LIGHT MICROSCOPY AND ELECTRON MICROSCOPY Paul Sims, Ralph Victoria Introduction Early Correlative Microscopy Early 4D Microscopy Correlative Light Microscope/Electron Microscope Today Light Microscope and Electron Microscope Have Different Requirements Finding the Same Cell Structure in Two Different Types of Microscope: Light Microscope/Scanning Electron Microscope Finding the Same Cell Structure in Two Different Types of Microscope: Light Microscope/Transmission Electron Microscope Cryo-Immobilization Followed by Post-Embedding Confocal Laser Scanning Microscopy on Thin Sections Tiled Montage Transmission Electron Microscope Images Aid Correlation Conclusion Metadata Structure Digital Rights Management Future Prospects CHAPTER BIOFILMS — SPATIOTEMPORAL R.J. Palmer, Jr., Janus A.J. Haagensen, Thomas R. Claus Sternberg Introduction Sample Presentation Flowcells and Other Water-Immersible Lenses Upright Versus Inverted Microscopes Setup of a Flow Chamber System Setup Example Making Bacteria Fluorescent Fluorescent Proteins Stains Nucleic Acid Stains Live/Dead Stain Fluorescence In Situ Hybridization General Procedure for Embedding of Flowcell-Grown Biofilms Antibodies Preparation of Labeled Primary Antibodies Imaging Bacteria Without Fluorescence Imaging Extracellular Polymeric Substances in Biofilms Application of Two-Photon Laser-Scanning Microscopy for Limitations of Confocal Laser Scanning Microscopy and Two-Photon Laser-Scanning Microscopy in Biofilm Temporal Experiments Time-Lapse Confocal Imaging Summary and Future Directions CHAPTER THREE-DIMENSIONAL MICROSCOPICAL IMAGES IN BIOLOGY Steffen Introduction Data and Metadata Management in Microscopes Recent Developments Image Information Management The Aims of Modern Microscope System Design Instrument Database Model System Requirements Image Database Model Selected Projects Bioimage. 865 Biomedicai Scientific Image DataBase Other Projects Criteria and Requirements for Microscopy Databases User Interface Query by Content CHAPTER MICROSCOPY Robert H. Webb A. Book and Review Articles B. Historical Interest С D. Technical E. General F. Adaptive Optics G. Differential H. Display I. Fiber-Optic Confocal Microscopes J. Index Mismatch K. Multiplex L. Nonlinear M. Polarization N. O. Point Spread Function P. Pupil Engineering Q. Thickness R. Turbidity S. Variants on the Main Theme xxiv APPENDIX TWO-PHOTON MICROSCOPY Mark B. and Christian Soeller Introduction Laser Safety Laser Alignment Testing Alignment and System Performance Laser Settings and Operation Monitoring Laser Performance Power Levels and Trouble-Shooting Choice of Pulse Length Controlling Laser Power Am I Seeing Two-Photon Excited Fluorescence or Stray Light and Non-Descanned Detection Laser Power Adjustment for Imaging at Depth Simultaneous Imaging of Multiple Labels Minimize Exposure During Orientation and Parameter Setting Ultraviolet-Excited Fluorochromes APPENDIX CURRENT COMMERCIAL CONFOCAL LIGHT MICROSCOPES USED IN BIOLOGY James B. Pawley Introduction BD-CARVII LaVision-BioTec TriM-Scope Leica Nikon Clsi Olympus Fluoview 1000-DSU Visitech VT Infmity-VT-eye Yokogawa Zeiss LSM-5-LIVE Fast Slit Scanner-LSM META-FCS APPENDIX TO KNOW ABOUT CHARGE-COUPLED DEVICES James B. Pawley Introduction Part Charge Coupling Readout Methods What Could Go Wrong? Quantum Efficiency Edge Effects Charge Loss Leakage or "Dark Charge" Blooming Incomplete Charge Transfer Charge Amplifiers What Is a Charge Amplifier? FET Noise Sources in the Charge-Coupled Device Fixed Pattern Noise Noise from the Charge Amplifier Where Is Zero? A New Idea: The Gain Register Amplifier!! Of Course, There Is One Snag! Part II: Evaluating a Charge-Coupled Device A. Important Charge-Coupled Device Specs for Live-Cell Stuff! B. Things That Are (Almost!) Irrelevant When Choosing a Charge-Coupled Device for Live-Cell Microscopy C. A Test You Can Do Yourself!!! D. Intensified Charge-Coupled Devices Index
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spelling	Handbook of biological confocal microscopy ed. James B. Pawley 3. ed. New York, NY Springer 2006 XXVIII, 985 S. zahlr. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Microscopia (métodos;técnicas) larpcal Microscopie confocale Terceira dimensão larpcal Confocal microscopy Imaging, Three-Dimensional Microscopy, Confocal Biowissenschaften (DE-588)4129772-6 gnd rswk-swf Konfokale Mikroskopie (DE-588)4336446-9 gnd rswk-swf Biologie (DE-588)4006851-1 gnd rswk-swf Mikroskopie (DE-588)4039238-7 gnd rswk-swf (DE-588)1071861417 Konferenzschrift 1989 San Antonio Tex. gnd-content Mikroskopie (DE-588)4039238-7 s Biologie (DE-588)4006851-1 s DE-604 Konfokale Mikroskopie (DE-588)4336446-9 s Biowissenschaften (DE-588)4129772-6 s Pawley, James B. Sonstige oth Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014196157&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Handbook of biological confocal microscopy Microscopia (métodos;técnicas) larpcal Microscopie confocale Terceira dimensão larpcal Confocal microscopy Imaging, Three-Dimensional Microscopy, Confocal Biowissenschaften (DE-588)4129772-6 gnd Konfokale Mikroskopie (DE-588)4336446-9 gnd Biologie (DE-588)4006851-1 gnd Mikroskopie (DE-588)4039238-7 gnd
subject_GND	(DE-588)4129772-6 (DE-588)4336446-9 (DE-588)4006851-1 (DE-588)4039238-7 (DE-588)1071861417
title	Handbook of biological confocal microscopy
title_auth	Handbook of biological confocal microscopy
title_exact_search	Handbook of biological confocal microscopy
title_exact_search_txtP	Handbook of biological confocal microscopy
title_full	Handbook of biological confocal microscopy ed. James B. Pawley
title_fullStr	Handbook of biological confocal microscopy ed. James B. Pawley
title_full_unstemmed	Handbook of biological confocal microscopy ed. James B. Pawley
title_short	Handbook of biological confocal microscopy
title_sort	handbook of biological confocal microscopy
topic	Microscopia (métodos;técnicas) larpcal Microscopie confocale Terceira dimensão larpcal Confocal microscopy Imaging, Three-Dimensional Microscopy, Confocal Biowissenschaften (DE-588)4129772-6 gnd Konfokale Mikroskopie (DE-588)4336446-9 gnd Biologie (DE-588)4006851-1 gnd Mikroskopie (DE-588)4039238-7 gnd
topic_facet	Microscopia (métodos;técnicas) Microscopie confocale Terceira dimensão Confocal microscopy Imaging, Three-Dimensional Microscopy, Confocal Biowissenschaften Konfokale Mikroskopie Biologie Mikroskopie Konferenzschrift 1989 San Antonio Tex.
url	http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014196157&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT pawleyjamesb handbookofbiologicalconfocalmicroscopy

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