Fundamentals of Light Microscopy and Electronic Imaging

ISBN-10: 047169214X

ISBN-13: 9780471692140

Edition: 2nd 2013

Authors: Douglas B. Murphy, Michael W. Davidson

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This book provides a coherent introduction to the principles and applications of the integrated electron microscope system. It 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. This revision includes new chapters on live cell imaging, measurement of protein dynamics, deconvolution microscopy, and interference microscopy.
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Book details

List price: $132.00
Edition: 2nd
Copyright year: 2013
Publisher: John Wiley & Sons, Limited
Publication date: 10/19/2012
Binding: Hardcover
Pages: 552
Size: 7.00" wide x 10.00" long x 1.25" tall
Weight: 3.102
Language: English

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