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| Preface | |
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| Acknowledgments | |
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| Fundamentals of Light Microscopy | |
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| Overview | |
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| Optical Components of the Light Microscope | |
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| Aperture and Image Planes in a Focused, Adjusted Microscope | |
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| Note: Objectives, Eyepieces, and Eyepiece Telescopes | |
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| Koehler Illumination | |
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| Adjusting the Microscope for Koehler Illumination | |
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| Note: Summary of Steps for Koehler Illumination | |
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| Note: Focusing Oil Immersion Objectives | |
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| Fixed Tube Length versus Infinity Optical Systems | |
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| Precautions for Handling Optical Equipment | |
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| Care and Maintenance of the Microscope | |
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| Exercise: Calibration of Magnification | |
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| Light and Color | |
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| Overview | |
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| Light as a Probe of Matter | |
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| The Dual Particle- and Wave-Like Nature of Light | |
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| The Quality of Light | |
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| Properties of Light Perceived by the Eye | |
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| Physical Basis for Visual Perception and Color | |
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| Addition and Subtraction Colors | |
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| Exercise: Complementary Colors | |
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| Illuminators, Filters, and the Isolation of Specific Wavelengths | |
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| Overview | |
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| Illuminators and Their Spectra | |
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| Illuminator Alignment and Bulb Replacement | |
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| Demonstration: Spectra of Common Light Sources | |
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| Demonstration: Aligning a 100-W Mercury Arc Lamp in an Epi-Illuminator | |
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| Filters for Adjusting the Intensity and Wavelength of Illumination | |
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| Effects of Light on Living Cells | |
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| Lenses and Geometrical Optics | |
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| Overview | |
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| Reflection and Refraction of Light | |
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| Image Formation by a Simple Lens | |
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| Note: Real and Virtual Images | |
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| Rules of Ray Tracing for a Simple Lens | |
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| Object-Image Math | |
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| The Principal Aberrations of Lenses | |
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| Designs and Specifications of Objectives | |
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| Condensers | |
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| Oculars | |
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| Microscope Slides and Coverslips | |
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| The Care and Cleaning of Optics | |
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| Exercise: Constructing and Testing an Optical Bench Microscope | |
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| Diffraction and Interference in Image Formation | |
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| Overview | |
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| Diffraction and Interference | |
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| The Diffraction Image of a Point Source of Light | |
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| The Constancy of Optical Path Length between Object and Image | |
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| Demonstration: Viewing the Airy Disk with a Pinhole Aperture | |
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| Effect of Aperture Angle on Diffraction Spot Size | |
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| Diffraction by a Grating and Calculation of Its Line Spacing, D | |
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| Demonstration: The Diffraction Grating | |
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| Abb�'s Theory for Image Formation in the Microscope | |
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| A Diffraction Pattern Is Formed in the Rear Aperture of the Objective | |
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| Demonstration: Observing the Diffraction Image in the Rear Focal Plane of a Lens | |
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| Preservation of Coherence: Essential Requirement for Image Formation | |
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| Exercise: Diffraction by Microscope Specimens | |
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| Diffraction and Spatial Resolution | |
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| Overview | |
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| Numerical Aperture | |
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| Spatial Resolution | |
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| Depth of Field and Depth of Focus | |
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| Optimizing the Microscope Image: A Compromise between Spatial Resolution and Contrast | |
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| Exercise: Resolution of Striae in Diatoms | |
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| Phase Contrast Microscopy and Darkfield Microscopy | |
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| Overview | |
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| Phase Contrast Microscopy | |
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| The Behavior of Waves from Phase Objects in Brightfield Microscopy | |
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| Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry | |
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| Darkfield Microscopy | |
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| Exercise: Darkfield Microscopy | |
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| Properties of Polarized Light | |
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| Overview | |
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| The Generation of Polarized Light | |
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| Demonstration: Producing Polarized Light with a Polaroid Filter | |
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| Polarization by Reflection and Scattering | |
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| Vectorial Analysis of Polarized Light Using a Dichroic Filter | |
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| Double Refraction in Crystals | |
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| Demonstration: Double Refraction by a Calcite Crystal | |
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| Kinds of Birefringence | |
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| Propagation of O and E Wavefronts in a Birefringent Crystal | |
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| Birefringence in Biological Specimens | |
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| Generation of Elliptically Polarized Light by Birefringent Specimens | |
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| Polarization Microscopy | |
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| Overview | |
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| Optics of the Polarizing Microscope | |
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| Adjusting the Polarizing Microscope | |
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| Appearance of Birefringent Objects in Polarized Light | |
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| Principles of Action of Retardation Plates and Three Popular Compensators | |
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| Demonstration: Making a �-Plate from a Piece of Cellophane | |
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| Exercise: Determination of Molecular Organization in Biological Structures Using a Full Wave Plate Compensator | |
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| Differential Interference Contrast Microscopy and Modulation Contrast Microscopy | |
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| Overview | |
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| The DIC Optical System | |
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| Demonstration: The Action of a Wollaston Prism in Polarized Light | |
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| Modulation Contrast Microscopy | |
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| Exercise: DIC Microscopy | |
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| Fluorescence Microscopy | |
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| Overview | |
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| Applications of Fluorescence Microscopy | |
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| Physical Basis of Fluorescence | |
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| Properties of Fluorescent Dyes | |
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| Demonstration: Fluorescence of Chlorophyll and Fluorescein | |
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| Autofluorescence of Endogenous Molecules | |
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| Demonstration: Fluorescence of Biological Materials under UV Light | |
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| Fluorescent Dyes and Proteins in Fluorescence Microscopy | |
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| Arrangement of Filters and the Epi-Illuminator in the Fluorescence Microscope | |
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| Objectives and Spatial Resolution in Fluorescence Microscopy | |
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| Causes of High Fluorescence Background | |
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| The Problem of Bleedthrough with Multiply Stained Specimens | |
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| Quenching, Blinking, and Photobleaching | |
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| Examining Fluorescent Molecules in Living Cells | |
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| Fluorescence Imaging of Dynamic Molecular Processes | |
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| Overview | |
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| Modes of Dynamic Fluorescence Imaging | |
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| F�rster Resonance Energy Transfer | |
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| Applications | |
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| Fluorescence Recovery after Photobleaching | |
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| TIRF Microscopy: Excitation by an Evanescent Wave | |
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| Advanced and Emerging Dynamic Fluoresence Techniques | |
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| Confocal Laser Scanning Microscopy | |
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| Overview | |
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| The Optical Principle of Confocal Imaging | |
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| Demonstration: Isolation of Focal Plane Signals with a Confocal Pinhole | |
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| Advantages of CLSM over Widefield Fluorescence Systems | |
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| Criteria Defining Image Quality and the Performance of an Electronic Imaging System | |
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| Confocal Adjustments and Their Effects on Imaging | |
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| Photobleaching | |
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| General Procedure for Acquiring a Confocal Image | |
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| Performance Check of a Confocal System | |
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| Fast (Real-Time) Imaging in Confocal Microscopy | |
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| Spectral Analysis: A Valuable Enhancement for Confocal Imaging | |
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| Optical Sectioning by Structured Illumination | |
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| Deconvolution Microscopy | |
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| Exercise: Effect of Confocal Variables on Image Quality | |
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| Two-Photon Excitation Fluorescence Microscopy | |
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| Overview | |
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| The Problem of Photon Scattering in Deep Tissue Imaging | |
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| Two-Photon Excitation Is a Nonlinear Process | |
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| Localization of Excitation | |
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| Why Two-Photon Imaging Works | |
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| Resolution | |
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| Equipment | |
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| Three-Photon Excitation | |
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| Second Harmonic Generation Microscopy | |
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| Superresolution Imaging | |
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| Overview | |
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| The RESOLFT Concept | |
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| Single-Molecule Localization Microscopy | |
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| Structured Illumination Microscopy | |
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| Stimulated Emission Depletion (STED) Microscopy: Superresolution by PSF Engineering | |
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| Imaging Living Cells with the Microscope | |
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| Overview | |
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| Labeling Strategies for Live-Cell Imaging | |
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| Control of Illumination | |
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| Control of Environmental Conditions | |
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| Optics, Detectors, and Hardware | |
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| Evaluating Live-Cell Imaging Results | |
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| Exercise: Fluorescence Microscopy of Living Tissue Culture Cells | |
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| Fundamentals of Digital Imaging | |
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| Overview | |
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| The Charge-Coupled Device (CCD Imager) | |
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| CCD Designs | |
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| Note: Interline CCD Imagers: The Design of Choice for Biomedical Imaging | |
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| Back-Thinned Sensors | |
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| EMCCD Cameras: High Performance Design for Greatest Sensitivity | |
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| Scientific CMOS: The Next Generation of Scientific Imagers | |
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| Camera Variables Affecting CCD Readout and Image Quality | |
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| Six Terms Define Imaging Performance | |
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| Aliasing | |
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| Color Cameras | |
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| Exercise: Evaluating the Performance of a CCD Camera | |
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| Digital Image Processing | |
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| Overview | |
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| Preliminaries: Image Display and Data Types | |
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| Histogram Adjustment | |
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| Adjusting Gamma (�) to Create Exponential LUTs | |
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| Flat-Field Correction | |
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| Image Processing With Filters | |
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| Signal-to-Noise Ratio | |
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| The Use of Color | |
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| Images as Research Data and Requirements for Scientific Publication | |
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| Exercise: Flat-Field Correction and Determination of S/N Ratio | |
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| Answer Key to Exercises | |
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| Materials for Demonstrations and Exercises | |
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| Sources of Materials for Demonstrations and Exercises | |
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| Glossary | |
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| Microscopy Web Resources | |
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| Recommended Reading | |
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| References | |
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| Index | |