نام كتاب : FUNDAMENTALS OF LIGHT MICROSCOPY AND ELECTRONIC IMAGINGنويسنده :Douglas B. Murphyانتشارات : JOHN WILEY & SONS-2001تعداد صفحات : 372 صفحهحجم فايل : 4.51 مگابايتفصول كتاب:1. FUNDAMENTALS OF LIGHT MICROSCOPY 1Overview 1Optical Components of the Light Microscope 1Note: Inverted Microscope Designs 3Aperture and Image Planes in a Focused, Adjusted Microscope 4Note: Using an Eyepiece Telescope to View the Objective Back Aperture 5Koehler Illumination 6Adjusting the Microscope for Koehler Illumination 7Note: Summary of Steps for Koehler Illumination 7Note: Focusing Oil Immersion Objectives 11Precautions for Handling Optical Equipment 11Exercise: Calibration of Magnification 122. LIGHT AND COLOR 15Overview 15Light as a Probe of Matter 15Light as Particles and Waves 18The Quality of Light 20Properties of Light Perceived by the Eye 21Physical Basis for Visual Perception and Color 22Positive and Negative Colors 24Exercise: Complementary Colors 263. ILLUMINATORS, FILTERS, AND ISOLATIONOF SPECIFIC WAVELENGTHS 29Overview 29Illuminators and Their Spectra 29Demonstration: Spectra of Common Light Sources 33Illuminator Alignment and Bulb Replacement 34Demonstration: Aligning a 100 W Mercury Arc Lamp in an Epi-illuminator 35“First On—Last Off ”: Essential Rule for Arc Lamp Power Supplies 36Filters for Adjusting the Intensity and Wavelength of Illumination 37Effects of Light on Living Cells 414. LENSES AND GEOMETRICAL OPTICS 43Overview 43Image Formation by a Simple Lens 43Note: Real and Virtual Images 45Rules of Ray Tracing for a Simple Lens 46Object-Image Math 46The Principal Aberrations of Lenses 50Designs and Specifications of Objective Lenses 53Condensers 56Oculars 56Microscope Slides and Coverslips 57The Care and Cleaning of Optics 58Exercise: Constructing and Testing an Optical Bench Microscope 595. DIFFRACTION AND INTERFERENCEIN IMAGE FORMATION 61Overview 61Defining Diffraction and Interference 61The Diffraction Image of a Point Source of Light 64Demonstration: Viewing the Airy Disk with a Pinhole Aperture 66Constancy of Optical Path Length Between the Object and the Image 68Effect of Aperture Angle on Diffraction Spot Size 69Diffraction by a Grating and Calculation of Its Line Spacing, d 71Demonstration: The Diffraction Grating 75Abbe’s Theory for Image Formation in the Microscope 77Diffraction Pattern Formation in the Back Aperture of the Objective Lens 80Demonstration: Observing the Diffraction Image in the Back FocalPlane of a Lens 81Preservation of coherence: An Essential Requirement for Image Formation 82Exercise: Diffraction by Microscope Specimens 846. DIFFRACTION AND SPATIAL RESOLUTION 85Overview 85Numerical Aperture 85Spatial Resolution 87Depth of Field and Depth of Focus 90Optimizing the Microscope Image: A Compromise Between SpatialResolution and Contrast 91Exercise: Resolution of Striae in Diatoms 937. PHASE CONTRAST MICROSCOPYAND DARK-FIELD MICROSCOPY 97Overview 97Phase Contrast Microscopy 97The Behavior of Waves from Phase Objects in Bright-Field Microscopy 99The Role of Differences in Optical Path Lengths 103The Optical Design of the Phase Contrast Microscope 103Alignment 106Interpretating the Phase Contrast Image 106Exercise: Determination of the Intracellular Concentration of Hemoglobin inErythrocytes by Phase Immersion Refractometry 110Dark-Field Microscopy 112Theory and Optics 112Image Interpretation 115Exercise: Dark-Field Microscopy 1168. PROPERTIES OF POLARIZED LIGHT 117Overview 117The Generation of Polarized Light 117Demonstration: Producing Polarized Light with a Polaroid Filter 119Polarization by Reflection and Scattering 121Vectorial Analysis of Polarized Light Using a Dichroic Filter 121Double Refraction in Crystals 124Demonstration: Double Refraction by a Calcite Crystal 126Kinds of Birefringence 127Propagation of O and E Wavefronts in a Birefringent Crystal 128Birefringence in Biological Specimens 130Generation of Elliptically Polarized Light by Birefringent Specimens 1319. POLARIZATION MICROSCOPY 135Overview 135Optics of the Polarizing Microscope 136Adjusting the Polarizing Microscope 138Appearance of Birefingent Objects in Polarized Light 139Principles of Action of Retardation Platesand Three Popular Compensators 139Demonstration: Making a Plate from a Piece of Cellophane 143Exercise: Determination of Molecular Organization in Biological StructuresUsing a Full Wave Plate Compensator 14810. DIFFERENTIAL INTERFERENCE CONTRAST (DIC)MICROSCOPY AND MODULATION CONTRASTMICROSCOPY 153Overview 153The DIC Optical System 153DIC Equipment and Optics 155The DIC Prism 157Demonstration: The Action of a Wollaston Prism in Polarized Light 158Formation of the DIC Image 159Interference Between O and E Wavefrontsand the Application of Bias Retardation 160Alignment of DIC Components 161Image Interpretation 166The Use of Compensators in DIC Microscopy 167Comparison of DIC and Phase Contrast Optics 168Modulation Contrast Microscopy 168Contrast Methods Using Oblique Illumination 169Alignment of the Modulation Contrast Microscope 172Exercise: DIC Microscopy 17311. FLUORESCENCE MICROSCOPY 177Overview 177Applications of Fluorescence Microscopy 178Physical Basis of Fluorescence 179Properties of Fluorescent Dyes 182Demonstration: Fluorescence of Chlorophyll and Fluorescein 183Autofluorescence of Endogenous Molecules 185Demonstration: Fluorescence of Biological MaterialsUnder Ultraviolet Light 189Arrangement of Filters and the Epi-illuminatorin the Fluorescence Microscope 189Objective Lenses and Spatial Resolution in Fluorescence Microscopy 194Causes of High-Fluorescence Background 196The Problem of Bleed-Through with Multiply Stained Specimens 197Examining Fluorescent Molecules in Living Cells 198Exercise: Fluorescence Microscopy of Living Tissue Culture Cells 19912. CONFOCAL LASER SCANNING MICROSCOPY 205Overview 205The Optical Principle of Confocal Imaging 208Demonstration: Isolation of Focal Plane Signalswith a Confocal Pinhole 211Advantages of CLSM Over Wide-Field Fluorescence Systems 213Criteria Defining Image Quality and the Performanceof an Electronic Imaging System 215Electronic Adjustments and Considerationsfor Confocal Fluorescence Imaging 217Photobleaching 223General Procedure for Acquiring a Confocal Image 224Two-Photon and Multi-Photon Laser Scanning Microscopy 226Confocal Imaging with a Spinning Nipkow Disk 229Exercise: Effect of Confocal Variables on Image Quality 23013. VIDEO MICROSCOPY 233Overview 233Applications and Specimens Suitable for Video 233Configuration of a Video Camera System 234Types of Video Cameras 236Electronic Camera Controls 238Demonstration: Procedure for Adjusting the Light Intensityof the Video Camera and TV Monitor 241Video Enhancement of Image Contrast 242Criteria Used to Define Video Imaging Performance 245Aliasing 249Digital Image Processors 249Image Intensifiers 250VCRs 251Systems Analysis of a Video Imaging System 252Daisy Chaining a Number of Signal-Handling Devices 254Exercise: Contrast Adjustment and Time-Lapse Recordingwith a Video Camera 25514. DIGITAL CCD MICROSCOPY 259Overview 259The Charge-Coupled Device (CCD Imager) 260CCD Architectures 267Note: Interline CCDs for Biomedical Imaging 268Analogue and Digital CCD Cameras 269Camera Acquisition Parameters Affecting CCD Readoutand Image Quality 269Imaging Performance of a CCD Detector 271Benefits of Digital CCD Cameras 276Requirements and Demands of Digital CCD Imaging 276Color Cameras 277Points to Consider When Choosing a Camera 278Exercise: Evaluating the Performance of a CCD Camera 27915. DIGITAL IMAGE PROCESSING 283Overview 283Preliminaries: Image Display and Data Types 284Histogram Adjustment 285Adjusting Gamma () to Create Exponential LUTs 287Flat-Field Correction 289Image Processing with Filters 292Signal-to-Noise Ratio 299Exercise: Flat-Field Correction and Determination of S/N Ratio 30516. IMAGE PROCESSING FOR SCIENTIFICPUBLICATION 307Overview 307Image Processing: One Variable Out of Many Affecting the Appearanceof the Microscope Image 307The Need for Image Processing 309Varying Processing Standards 309Record Keeping During Image Acquisition and Processing 310Note: Guidelines for Image Acquisition and Processing 310Use of Color in Prints and Image Displays 312Colocalization of Two Signals Using Pseudocolor 313A Checklist for Evaluating Image Quality 315