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Introduction to Optical Microscopy,
Digital Imaging, and Photomicrography

This treatise on optical microscopy is divided into several sections that are available through the links displayed immediately to the left (in the darker navigational boxes) and below. In order to print the entire microscopy primer as a paper document, you must download each link independently, send the file to your printer, and put the results together.

In the Bibliography, we have included links to other works on optical microscopy and our section on Web Resources contains links to other microscopy sites on the Internet. This material is targeted for educational purposes only, and is not available to be posted on remote websites (either commercial or educational) or distributed in any electronic format.

Frequently Asked Questions - Mortimer Abramowitz, senior microscopist at Olympus America Inc., answers the 50 most commonly asked questions about microscopy and photomicrography.

Physics of Light and Color - Visible light represents only a small portion of the entire electromagnetic spectrum of radiation that extends from high-frequency gamma rays through X-rays, ultraviolet light, infrared radiation and microwaves to very low frequency long-wavelength radio waves. The complex phenomenon of visible light is classically discussed in terms of rays and wavefronts. Starting with the nature of electromagnetic radiation, a wide variety of topics are covered in this section, including refraction, reflection, diffraction, interference, birefringence, polarization, primary colors, human vision, mirrors, prisms, beamsplitters, laser systems, geometrical optics, filtration, color temperature, and the speed of light.

Anatomy of the Microscope - A thorough discussion of the elements that comprise modern microscopes and theories behind important concepts such as magnification, image formation, objective specifications, Köhler illumination, optical aberrations, immersion media, light sources, eyepieces, condensers, and ergonomics, among others.

Light Sources for Optical Microscopy - The performance of the various illumination sources available for optical microscopy depends on the emission characteristics and geometry of the source, as well as the focal length, magnification and numerical aperture of the collector lens system. In gauging the suitability of a particular light source, the important parameters are structure (the spatial distribution of light, source geometry, coherence, and alignment), the wavelength distribution, spatial and temporal stability, brightness, and to what degree these various parameters can be controlled.

Specialized Microscopy Techniques - More advanced topics in microscopy are described in this section, including contrast enhancement techniques, such as Hoffman modulation and oblique illumination, as well as fluorescence microscopy, differential interference contrast, phase contrast and other important optical techniques used in microscopy.

Digital Imaging in Optical Microscopy - Digitization of a video or electronic image captured through an optical microscope results in a dramatic increase in the ability to enhance features, extract information, or modify the image. When compared to the traditional mechanism of image capture, photomicrography on film, digital imaging and post-acquisition processing enables a reversible, essentialy noise-free modification of the image as an ordered matrix of integers rather than a series of analog variations in color and intensity. This section addresses a variety of current topics in image acquistion and processing for optical microscopy.

Photomicrography - The primary medium for photomicrography was film until the past decade when improvements in electronic cameras and computer technology made digital imaging cheaper and easier to use than conventional photography. This section addresses the classical methods of photomicrography on film and includes a comprehensive analysis of the cause and correction of errors and faults in photomicrography.

Concepts and Formulas in Microscopy - Many of the basic concepts in optical microscopy can be distilled into a few rules and formulas. This section, from the Nikon MicroscopyU website, reviews the important elements and equations necessary for an understanding of concepts such as resolution, field of view, numerical aperture, image brightness, useful magnification range, and depth of field. Many of these topics are discussed in much greater detail in the Molecular Expressions Microscopy Primer, but this should serve as a quick reference to interested visitors.

Fundamentals of Stereomicroscopy - Considering the wide range of accessories currently available for stereomicroscope systems, this class of instruments is extremely useful in a multitude of applications. Stands and illuminating bases for a variety of contrast enhancement techniques are available from all of the manufacturers, and can be adapted to virtually any working situation. There are a wide choice of objectives and eyepieces, enhanced with attachment lenses and coaxial illuminators that are fitted to the microscope as an intermediate tube. Working distances can range from 3-5 centimeters to as much as 20 centimeters in some models, allowing for a considerable amount of working room between the objective and specimen.

Virtual Microscopy - Our interactive Java-powered virtual microscopes allow visitors to explore selected samples with their Web browser using a variety of contrast-enhancing techniques. The virtual microscope techniques include differential interference contrast, fluorescence, Rheinberg illumination, and polarized light.

Olympus FluoView Laser Scanning Confocal Microscopy - The new Olympus FluoViewTM FV1000 is the latest in point-scanning, point-detection, confocal laser scanning microscopes designed for today's intensive and demanding biological research investigations. Excellent resolution, bright and crisp optics, and high efficiency of excitation, coupled to an intuitive user interface and affordability are key characteristics of this state-of-the-art optical microscopy system.

The Olympus MIC-D Digital Microscope - Olympus has thrown the doors open to a new era in optical microscopy education with the introduction of the MIC-D inverted digital microscope. Designed specifically for a wide spectrum of applications ranging from basic classroom instruction to more advanced laboratory analysis, this versatile microscope features a palette of contrast enhancing techniques that rival many research-level instruments.

Java and Flash Tutorial Basics - Interactive Java and Flash tutorials have been developed to help students explore complex concepts in all phases of optical microscopy, the physics of light and color, photomicrography, and digital imaging technology. The tutorials are embedded within web pages that contain accompanying discussions about the subject phenomenon and instructions for use and control of the applets. This section discusses the basics of tutorial navigation and operation.

Museum of Microscopy - Visit our museum of microscopes ranging from ancient sixteenth-century single-lens Dutch models to modern multipurpose microprocessor-powered research microscopes. These microscopes were constructed in 3D Studio Max to recreate how they might have appeared when first built.

Innovations in Light Microscopy - Noted microscopist and author Martin L. Scott examines recent advances in the microscope optical train, including infinity-corrected optical systems and new design motifs for modern objectives. Also included in the article is an overview of how these innovations have led to better images when coupled with the latest state-of-the-art techniques in optical microscopy. Ergonomics, a growing concern of microscope users and manufacturers, is also discussed.

Microscope: Basics and Beyond (50 pages in PDF format; 20.7 Mbytes) - Download the latest edition of Mortimer Abramowitz's renowned introduction to optical microscopy in full color. The volume covers all of the important basic concepts, ranging from simple magnifiers to complex compound microscopes, including illumination, objectives, eyepieces, condensers, aberration, Köhler illumination, resolution, numerical aperture, and depth of field. Numerous appendices review focusing of the microscope and oil immersion, and contain useful numbers, formulas, and a short bibliography.

Optical Microscopy Review - Download our latest review article on optical microscopy. Co-authored by Michael W. Davidson and Mortimer Abramowitz, this article discusses the basics of image formation, objectives, eyepieces, condensers, contrast enhancement, and photomicrography with full-color illustrations. Click on the link to download the document in PDF format (1.75 Mb).

Recommended Books on Optical Microscopy - Several hundred books dealing with various aspects of optical microscopy and related fields are currently available from the booksellers. This section lists the Molecular Expressions website team top 10 recommended books on microscopy, digital imaging, fluorescence, video, and microtechnique.

Recommended Books on Confocal Microscopy - A surprisingly limited number of books dealing with various aspects of laser scanning and spinning disk confocal microscopy and related techniques are currently available from the booksellers. This section lists the FluoView Resource Center website development team's top 12 recommended books. Although the volumes listed in this section deal pricipally with confocal microscopy and related methodology, there exist a number of additional books that contain focused treatments of the materials described below, and these should also be consulted for specific techniques and timely review articles.

ZEISS Campus Microscopy Reference Library - The scientific literature contains abundant resources in the form of books, review articles, and original research reports that deal with numerous topics in optical microscopy. The Carl Zeiss MicroImaging Online Campus Reference Library contains links to selected reports that should be useful to investigators seeking introductory material on a variety of techniques, probes, light sources, and live-cell imaging applications.

Web Resources - The website team has reviewed and provided links to over 100 microscopy websites in our resources section. The sites are arranged according to origin (university or commercial), target audience, and by educational level. Also included are links to glossaries and newsgroups.

Bibliography - All of the reference materials used in preparing the Molecular Expressions Optical Microscopy Primer are cited in this section along with other books about electron and scanning probe microscopy, photomicrography, highly specialized microscopy techniques, and older books dealing with the history of microscopy and microscopy in the early twentieth century.

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