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