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| Introduction: The Optical Microscope in Cell Biology | |
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| Contributors | |
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| Acknowledgments | |
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| The Light Microscope | |
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| Lenses and Microscopes | |
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| The Back Focal Plane of a Lens | |
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| Good Resolution | |
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| Resolution: Rayleigh's Approach | |
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| Abbe | |
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| Add a Drop of Oil | |
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| K�hler Illumination | |
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| References | |
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| Optical Contrasting Techniques | |
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| Darkfield | |
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| Phase Contrast | |
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| Polarization | |
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| Differential Interference Contrast | |
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| Hoffman Modulation Contrast | |
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| Which Technique Is Best? | |
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| References | |
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| Fluorescence and Fluorescence Microscopy | |
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| What Is Fluorescence? | |
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| What Makes a Molecule Fluorescent? | |
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| The Fluorescence Microscope | |
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| Optical Arrangement | |
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| Light Source | |
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| Filter Sets: Excitation Filter, Dichroic Mirror, and Barrier Filter | |
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| References | |
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| Image Capture | |
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| Optical Layout for Image Capture | |
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| Color Recording | |
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| Additive Color Model | |
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| Subtractive Color Model | |
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| CCD Cameras | |
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| Frame-Transfer Array | |
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| Interline-Transfer Array | |
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| Back Illumination | |
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| Binning | |
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| Capturing Color | |
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| Filter Wheels | |
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| Filter Mosaics | |
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| Three CCD Elements with Dichroic Beamsplitters | |
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| Boosting the Signal | |
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| References | |
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| The Confocal Microscope | |
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| The Scanning Optical Microscope | |
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| The Confocal Principle | |
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| Resolution and Point Spread Function | |
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| Lateral Resolution in the Confocal Microscope | |
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| Practical Confocal Microscopes | |
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| The Light Source: Lasers | |
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| Gas Lasers | |
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| Solid-State Lasers | |
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| Semiconductor Lasers | |
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| Supercontinuum Lasers | |
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| Laser Delivery | |
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| The Primary Beamsplitter | |
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| Beam Scanning | |
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| Pinhole and Signal Channel Configurations | |
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| Detectors | |
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| References | |
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| Further Reading | |
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| The Digital Image | |
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| Pixels and Voxels | |
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| Contrast | |
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| Spatial Sampling: The Nyquist Criterion | |
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| Temporal Sampling: Signal-to-Noise Ratio | |
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| Multichannel Images | |
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| References | |
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| Further Reading | |
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| Aberrations and Their Consequences | |
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| Geometrical Aberrations | |
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| Spherical Aberration | |
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| Coma | |
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| Astigmatism | |
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| Field Curvature | |
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| Chromatic Aberration | |
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| Chromatic Difference of Magnification | |
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| Practical Consequences | |
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| Apparent Depth | |
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| References | |
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| Further Reading | |
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| Nonlinear Microscopy | |
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| Multiphoton Microscopy | |
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| Principles of Two-Photon Fluorescence | |
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| Theory and Practice | |
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| Lasers for Nonlinear Microscopy | |
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| Advantages of Two-Photon Excitation | |
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| Construction of a Multiphoton Microscope | |
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| Fluorochromes for Multiphoton Microscopy | |
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| Second Harmonic Microscopy | |
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| Summary | |
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| References | |
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| Further Reading | |
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| High-Speed Confocal Microscopy | |
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| Tandem Scanning (Spinning Disk) Microscopes | |
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| Petr�n System | |
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| One-Sided Tandem Scanning Microscopes (OTSMS) | |
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| Microlens Array: The Yokogawa System | |
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| Slit-Scanning Microscopes | |
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| Multipoint-Array Scanners | |
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| Structured Illumination | |
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| References | |
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| Further Reading | |
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| Deconvolution and Image Processing | |
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| Deconvolution | |
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| Deconvolving Confocal Images | |
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| Image Processing | |
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| Grayscale Operations | |
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| Image Arithmetic | |
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| Convolution: Smoothing and Sharpening | |
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| References | |
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| Further-Reading | |
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| Three-Dimensional Imaging: Stereoscopy and Reconstruction | |
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| Surfaces: Two-and-a-Half Dimensions | |
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| Perception of the 3D World | |
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| Motion Parallax | |
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| Convergence and Focus of Our Eyes | |
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| Perspective | |
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| Concealment of One Object by Another | |
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| Our Knowledge of the Size and Shape of Everyday Things | |
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| Light and Shade | |
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| Limitations of Confocal Microscopy | |
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| Stereoscopy | |
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| Three-Dimensional Reconstruction | |
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| Techniques That Require Identification of "Objects" | |
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| Techniques That Create Views Directly from Intensity Data | |
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| Simple Projections | |
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| Weighted Projection (Alpha Blending) | |
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| References | |
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| Green Fluorescent Protein | |
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| Structure and Properties of GFP | |
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| GFP Variants | |
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| Applications of GFP | |
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| Heat Shock | |
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| Cationic Lipid Reagents | |
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| DEAE-Dextran and Polybrene | |
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| Calcium Phosphate Coprecipitation | |
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| Electroporation | |
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| Microinjection | |
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| Gene Gun | |
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| Plants: Agrobacterium | |
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| References | |
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| Fluorescent Staining | |
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| Immunolabeling | |
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| Types of Antibody | |
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| Raising Antibodies | |
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| Labeling | |
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| Fluorescent Stains for Cell Components and Compartments | |
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| References | |
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| Quantitative Fluorescence | |
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| Fluorescence Intensity Measurements | |
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| Linearity Calibration | |
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| Measurement | |
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| Colocalization | |
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| Ratio Imaging | |
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| Cell Loading | |
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| Membrane Potential | |
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| Fast-Response Dyes | |
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| Slow-Response Dyes | |
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| Fluorescence Recovery after Photobleaching | |
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| References | |
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| Advanced Fluorescence Techniques: FLIM, FRET, and FCS | |
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| Fluorescence Lifetime | |
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| Practical Lifetime Microscopy (FLIM) | |
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| Frequency Domain | |
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| Time Domain | |
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| Fluorescence Resonant Energy Transfer (FRET) | |
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| Why Use FRET? | |
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| Identifying and Quantifying FRET | |
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| Increase in Brightness of Acceptor Emission | |
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| Quenching of Emission from the Donor | |
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| Lifetime of Donor Emission | |
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| Protection from Bleaching of Donor | |
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| Fluorescence Correlation Spectroscopy (FCS) | |
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| Raster Image Correlation Spectroscopy | |
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| References | |
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| Further Reading | |
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| Evanescent Wave Microscopy | |
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| The Near-Field and Evanescent Waves | |
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| Total Internal Reflection Microscopy | |
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| Near-Field Microscopy | |
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| References | |
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| Beyond the Diffraction Limit | |
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| 4Pi and Multiple-Objective Microscopy | |
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| Stimulated Emission Depletion (STED) | |
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| Structured Illumination | |
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| Stochastic Techniques | |
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| Super-Resolution Summary | |
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| References | |
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| Microscope Care and Maintenance | |
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| Cleaning | |
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| The Fluorescent Illuminator | |
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| Keeping Cells Alive under the Microscope | |
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| Chambers | |
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| Light | |
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| Movement | |
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| Finally | |
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| Antibody Labeling of Plant and Animal Cells: Tips and Sample Schedules | |
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| Antibodies: Tips on Handling and Storage | |
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| Pipettes: Tips on Handling | |
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| Antibodies and Antibody Titrations | |
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| Example | |
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| Immunofluorescence Protocol | |
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| Method | |
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| Multiple Labeling and Different Samples | |
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| Plant Material | |
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| Protocol | |
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| Diagram Showing Position of Antibodies on Multiwell Slide273 | |
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| Image Processing with ImageJ | |
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| Introduction | |
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| Different Windows in ImageJ | |
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| Image Levels | |
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| Colors and Look-Up Tables | |
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| Size Calibration | |
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| Image Math | |
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| Quantification | |
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| Stacks and 3D Representation | |
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| FFT and Image Processing | |
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| Macro Language in ImageJ | |
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| Index | |