Computer Operation for Microscope Photometry
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Summary
Suitable for both microscopists seeking computer skills and PC enthusiasts interested in light microscopy, this interdisciplinary text explores the capabilities of the computer-assisted light microscope. Written in clear, simple language, the book explains how computer technology expands the usefulness of the light microscope in spectrophotometry, fluorometry, polarimetry, spatial scanning, and related fields.
Beginning with the basic features of light microscopy and personal computer interfacing, the text explains how to make photometric measurements and covers spectrophotometry, stepper motors, and server motors. Polarized light and video image analysis complete this introduction to the field. While software examples are provided to illustrate specific techniques, most operations are described as generalized algorithms that can be adapted to any appropriate high-level language, and used with almost any configuration of the microscope. The book suggests new experiments to inspire further study. Promising new areas of interest, such as the use of fluorescence and polarization, are also included.
Computers have radically changed the field of light microscopy in recent decades. Computer Operations for Microscope Photometry helps you master the new techniques.
Table of Contents
Introduction
Purpose of This Book
Power Supply
Stabilization
Arc Lamps
Illuminator
Adjustment
Assembly
Köhler Illumination
Lamp Size
Adjustments
Testing
Problems
Resolution
Numerical Aperture
Comparison with Electron Microscope
Microscope Objectives
Achromats
Cleaning
Enhanced Achromats
Apochromats
Fluorite Objectives
Polarized Light Objectives
Epi-Objectives
Phase Contrast
Differential Interference-Contrast
PC Interfacing
GPIB (HP-IB or IEEE 488)
RS-232
VXI
Software
Graphics
Different Types of Scanning
Dimensions of Scanning
Illuminators
Introduction
Arc Lamps
Historical
Short-Arc Mercury Lamp
Short-Arc Xenon Lamp
Tungsten-Halogen Filament Lamps
Emission Spectra
Control of Illumination Intensity
Shutters and Apertures
Introduction
Shutter and Aperture Locations
Illumination Shutter
Photometric Field Aperture
Ocular Shutter
Photometer Shutter
Photometric Measuring Aperture
Secondary Illuminator Shutter
Protective Shutter
Shutter-Photometer Interactions
Hardware
Software
PMT Response
Comparison of Two Shutters
Minimum Exposure Protocol
Shutter Software for Normal Operations
Power-Up and Power-Down
Measuring Position
Viewing Position
Visible Light Viewing Position for Ultraviolet Fluorometry
Standardization of Photometer for Fluorometry
Dark-Field
Dark-Field Plus Ambient
Negative Shutter Test
Positive Shutter Test
Diagnostics
Software
Importance of Timing
Photometry
Introduction
Analog-to-Digital Conversion
Dual-Slope Method
Successive Approximation Method
Nyquist Theorem
Photoresistive Detectors
Spectral Response
Photovoltaic Detectors
Solar Cell
Photomultipliers
Hardware
Software
Monitoring Performance
Different Controllers
Transient Events
Amplifier Gain
Good and Bad PMT Settings
Monochromators and Spectrophotometry
Introduction
Step-and-Measure
Data-Structure
Scanner Hierarchy
Spectral Scanning Algorithm
Monochromators
Software Options
Prism Monochromator
Grating Monochromator
Stray-Light Filters
Monochromator Location
Continuous Interference Filter
Software
Distributional Error
Mechanical Stages and Scanning
Introduction
Stepper Motors
Operational Problems
Software
Histochemical Mapping
Hardware
Software
Results
Switching Optical Fibers
Tilting Stages
Polarized Light
Introduction
Principal Axes
Types of Birefringence
Retardation
Interference Colors
Types of Compensators
de Sénarmot Compensator Algorithm
Brace- Köhler Compensator Algorithm
Knob Rotation Actuator
Operational Features
Continuous Measurement of Birefringence
Hardware
Software
Results and Practical Problems
Tilting Stage and Polarization of Reflected Light
Hardware
Theory
Illumination and Tilting Restraints
Extinction Coefficient
Intrinsic Anisotropy
Vertical 0° vs. Lateral 45° Illumination
Alignment and Depth of Focus
Surface Irregularities
Spectrophotometry of Tilted Standards
Spectrophotometry of Tissue Surface Reflectance
Spectrophotometry of Diffuse Reflectance
Polarimetry of Surface Reflectance
Iridescence
Why is the Polarized Light Microscope Seldom Used by Biologists?
Fluorescence
Introduction
Excitation vs. Emission
Dia-Excitation
Ultraviolet Safety
Epi-Excitation
Importance of Collagen Autofluorescence
Fluorometry Software
Relative Spectral Fluorescence Intensity
Spectrofluorometry
Comparison of Methods at High Light Intensity
Comparison of Methods at Low Light Intensity
High vs. Low Light Intensity for Standardization
Fluorescence Blank and Pseudofluorescence
Video
Introduction
Pixels and Gray Levels
Video Cameras
Software
Noise
Contrast Enhancement
Neighborhood Averaging Kernels
Laplacian Kernels
Erosion and Dilation
Summary
Photodiode Array Spectrography
Introduction
Advantage of PDA over Shutter Pulse
Mounting the PDA
PDA Spectrograph
CAM
Retrospective Standardization
Fluorometry of Collagen and Elastin
Sources of Variance: Samples vs. Photodiodes
Software for Rate of Quenching
Effect of pH and Measuring Direction
Liberating the Microscope with Fiberoptics
Introduction
Optical Fibers
Step-Index Fibers
Numerical Aperture
Monomode Fibers
Coupling
Fiber Arrangements
Internal Reflectance in Tissues
Remote Spectrophotometry
Standardization
Examples
Spatial Scanning
Monochromatic Scattering with a Laser
Polychromatic Scattering with a CAM
Goniospectrophotometry
Hardware
Standardization
Remote Fluorometry
Needle Penetration Fluorometry
Vignette Window Effect
Spectral Window Effect
Internal Reflections
Hypodermic Needle Angle
Widespread Occurrence of Aperture Effects
Coherent Fiberoptic Light Guide for Remote Imaging
Polarized Light
Transmittance
Back-Scattering
Programming the Sample Environment
Introduction
Regulating pH
Hardware
Software
Example
Refractive Index Control
Temperature Control
Temperature Sensor
Thermal Stage
Control Loops
Planning the Experimental Protocol
References
Index
