"This book will provide individuals without background knowledge in optical physics, electronics, or image processing with many of the basic facts they need to know to understand both the power and limitations of their images." Cell Biology Education on the First Edition Fundamentals of Light Microscopy and Electronic Imaging, Second Edition provides a coherent introduction to the principles and applications of the integrated optical microscope system, covering both theoretical and practical considerations. It expands and updates discussions of multi-spectral imaging, intensified digital cameras, signal colocalization, and uses of objectives, and offers guidance in the selection of microscopes and electronic cameras, as well as appropriate auxiliary optical systems and fluorescent tags. Written in simple, clear language, the book is divided into three sections covering optical principles in diffraction and image formation, basic modes of light microscopy, and components of modern electronic imaging systems and image processing operations. Each chapter introduces relevant theory, followed by descriptions of instrument alignment and image interpretation. Including new sections on live cell imaging, measurement of protein dynamics, deconvolution, multiphoton microscopy, and superresolution microscopy, Fundamentals of Light Microscopy and Electronic Imaging, Second Edition features the following chapters: * Fundamentals of Light Microscopy * Light and Color * Illuminators, Filters, and the Isolation of Specific Wavelengths * Lenses and Geometrical Optics * Diffraction and Interference in Image Formation * Diffraction and Spatial Resolution * Phase Contrast Microscopy and Darkfield Microscopy * Properties of Polarized Light * Polarization Microscopy * Differential Interference Contrast (DIC) Microscopy and Modulation Contrast Microscopy * Fluorescence Microscopy * Fluorescence Imaging of Dynamic Molecular Processes * Confocal Laser Scanning Microscopy * TwoPhoton Excitation Fluorescence Microscopy * Superresolution Imaging * Imaging Living Cells with the Microscope * Fundamentals of Digital Imaging * Digital Imaging Processing

InhaltsangabePreface xi Acknowledgments xii 1. FUNDAMENTALS OF LIGHT MICROSCOPY 1 Overview 1 Optical Components of the Light Microscope 1 Aperture and Image Planes in a Focused, Adjusted Microscope 5 Note: Objectives, Eyepieces, and Eyepiece Telescopes 6 Koehler Illumination 9 Adjusting the Microscope for Koehler Illumination 9 Note: Summary of Steps for Koehler Illumination 11 Note: Focusing Oil Immersion Objectives 14 Fixed Tube Length versus Infi nity Optical Systems 15 Precautions for Handling Optical Equipment 16 Care and Maintenance of the Microscope 17 Exercise: Calibration of Magnification 17 2. LIGHT AND COLOR 21 Overview 21 Light as a Probe of Matter 21 The Dual Particle- and Wave-Like Nature of Light 25 The Quality of Light 26 Properties of Light Perceived by the Eye 27 Physical Basis for Visual Perception and Color 28 Addition and Subtraction Colors 30 Exercise: Complementary Colors 32 3. ILLUMINATORS, FILTERS, AND THE ISOLATION OF SPECIFIC WAVELENGTHS 35 Overview 35 Illuminators and Their Spectra 35 Illuminator Alignment and Bulb Replacement 41 Demonstration: Spectra of Common Light Sources 41 Demonstration: Aligning a 100-W Mercury Arc Lamp in an Epi-Illuminator 43 Filters for Adjusting the Intensity and Wavelength of Illumination 45 Effects of Light on Living Cells 50 4. LENSES AND GEOMETRICAL OPTICS 53 Overview 53 Reflection and Refraction of Light 53 Image Formation by a Simple Lens 56 Note: Real and Virtual Images 57 Rules of Ray Tracing for a Simple Lens 58 ObjectImage Math 58 The Principal Aberrations of Lenses 62 Designs and Specifi cations of Objectives 65 Condensers 71 Oculars 72 Microscope Slides and Coverslips 73 The Care and Cleaning of Optics 73 Exercise: Constructing and Testing an Optical Bench Microscope 76 5. DIFFRACTION AND INTERFERENCE IN IMAGE FORMATION 79 Overview 79 Diffraction and Interference 80 The Diffraction Image of a Point Source of Light 83 The Constancy of Optical Path Length between Object and Image 85 Demonstration: Viewing the Airy Disk with a Pinhole Aperture 85 Effect of Aperture Angle on Diffraction Spot Size 87 Diffraction by a Grating and Calculation of Its Line Spacing, D 89 Demonstration: The Diffraction Grating 93 Abbé's Theory for Image Formation in the Microscope 94 A Diffraction Pattern Is Formed in the Rear Aperture of the Objective 97 Demonstration: Observing the Diffraction Image in the Rear Focal Plane of a Lens 98 Preservation of Coherence: Essential Requirement for Image Formation 99 Exercise: Diffraction by Microscope Specimens 101 6. DIFFRACTION AND SPATIAL RESOLUTION 103 Overview 103 Numerical Aperture 103 Spatial Resolution 105 Depth of Field and Depth of Focus 109 Optimizing the Microscope Image: A Compromise between Spatial Resolution and Contrast 109 Exercise: Resolution of Striae in Diatoms 112 7. PHASE CONTRAST MICROSCOPY AND DARKFIELD MICROSCOPY 115 Overview 115 Phase Contrast Microscopy 115 The Behavior of Waves from Phase Objects in Brightfi eld Microscopy 119 Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry 128 Darkfi eld Microscopy 129 Exercise: Darkfi eld Microscopy 133 8. PROPERTIES OF POLARIZED LIGHT 135 Overview 135 The Generation of Polarized Light 135 Demonstration: Producing Polarized Light with a Polaroid Filter 137 Polarization by Refl ection and Scattering 139 Vectorial Analysis of Polarized Light Using a Dichroic Filter 139 Double Refraction in Crystals 142 Demonstration: Double Refraction by a Calcite Crystal 144 Kinds of Birefringence 145 Propagation of O and E Wavefronts in a Birefringent Crystal 146 Birefringence in Biological Specimens 148 Generation of Elliptically Polarized Light by Birefringent Specimens 149 9. POLARI

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