Computer Operation for Microscope Photometry: 1st Edition (Hardback) book cover

Computer Operation for Microscope Photometry

1st Edition

By H. J. Swatland

CRC Press

256 pages

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Hardback: 9780849316975
pub: 1997-11-25
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Description

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

Subject Categories

BISAC Subject Codes/Headings:
SCI010000
SCIENCE / Biotechnology
SCI043000
SCIENCE / Research & Methodology
SCI047000
SCIENCE / Microscopes & Microscopy