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Interactive Java Tutorials

Specialized Microscopy Techniques

We have constructed a variety of Java tutorials designed to help students grasp the more esoteric features of specialized microscopy techniques. Please use the links below to visit the tutorials in our collection.

Background Intensity Effect on Contrast - Explore the effect of background intensity on image contrast in optical microscopy using this interactive Java tutorial. The tutorial plots image contrast as a function of image intensity and displays the contrast range for each background intensity selected by the slider. It becomes apparent after operating this tutorial that when background intensity is less than 0.1, small changes in image intensity produce large changes in contrast. As the background intensity is increased up to 1.0, changes in image intensity have a progressively smaller effect on overall contrast.

Optical Gradients in Phase Objects - This interactive tutorial investigates how light passes through a phase object whose sides are not plano-parallel but rather appear as a prism. Light passes through the hemispherically-shaped phase object from the bottom and travels through, exiting at the top on the rounded surface. For each point where the light ray passes through the curved phase upper surface, a tangent can be drawn to the surface to create a minute prism.

Optical Path Difference - Explore optical path differences for phase objects as a function of specimen and surrounding medium refractive index variations. The optical path difference is the product of two terms: the thickness (t) and the difference in refractive index (n). The OPD can often be quite large even though the thickness of the object is quite thin. On the other hand, when the refractive indices of the specimen and the surrounding medium are equal, the OPD is zero even if the specimen thickness is very large. In this case, light traveling through the object is merely delayed (a phase difference) relative to the light passing an equal thickness of the surround. Phase differences are not detectable by the human eye.

Integrated Circuit Inspection With Reflected Confocal Microscopy - Explore real-time confocal imaging of integrated circuits with reflected light confocal microscopy using this interactive Java tutorial. Choose from a wide spectrum of integrated circuits, including many of the latest microprocessors available for personal computers.

Reflected Light Microscopy - This tutorial examines the optical pathways in a reflected light microscope. The visitor can open and close the iris diaphragms controlling the condenser and the field lens.

Rheinberg Illumination - This interesting tutorial allows the student to adjust color values in both the oblique and central Rheinberg filters to demonstrate how these color changes affect specimen illumination.

Fluorescence Filter Cubes - Explore how various excitation and barrier fluorescence filter combinations can be used to probe fluorochromes at specific wavelengths.

Fluorescence Filter Spectra - Discover how fluorescence filters can be controlled to pass only certain wavelengths and how this affects the light that enters the microscope observation tube. The tutorial allows visitors to experiment with various filter combinations.

Reflected Light Fluorescence Microscopy - This tutorial examines the optical pathways in a reflected light fluorescence microscope. Filter cubes in the optical path can be adjusted to provide many different excitation and barrier filter combinations.

Microscope Assembly - Explore how a microscope is assembled by using this interactive Java tutorial to "build" a complex fluorescence microscope.

Spherulites Under Polarized Light - This tutorial examines how crystals with spherulitic growth habits appear under crossed-polarized illumination. The visitor can even add a quarter wavelength and retardation plate to the optical pathway.

Cardioid Condensers - This tutorial examines how light is refracted, diffracted, and scattered into the objective by a specimen when obliquely illuminated by a Cardioid condenser.

Differential Interference Contrast (DIC) - The DIC tutorial explores how changes in the Wollaston prism position affects how images are seen in the microscope viewfield. The tutorial also permits addition of a retardation plate to the optical pathway to add color and contrast to the specimen.

Hoffman Modulation Contrast - Hoffman modulation contrast is a technique used for increasing visibility and contrast, especially for unstained objects and living material. This tutorial explores how modulation contrast affects the image of a deer tick in the microscope viewfield.

Darkfield Microscopy

Light Cone Formation With Abbe Darkfield Condensers - This tutorial explores how a changes in the size of the opaque stop in a simple Abbe darkfield illumination condenser affects light cone shape and numerical aperture. As the size of the opaque stop is changed, the numerical aperture of the hollow light cone also changes to produce a new light cone of different numerical aperture. The condenser numerical aperture is given for each light cone along with the suggested objective magnification.

Darkfield Condenser Adjustment - Use this interactive tutorial to experiment with the adjustable parameters in condenser alignment and configuration for darkfield microscopy. Microscope adjustments include specimen focus, darkfield stop size and translation, and condenser height. We have even included a Bertrand lens to allow visitors to observe the darkfield stop within the objective rear focal plane.

Hollow Light Cone Numerical Aperture - Investigate how the shape and size of the hollow cone of light emitted by a hypothetical reflecting darkfield condenser changes with numerical aperture. The darkfield condenser utilized in the tutorial is a paraboloid-style condenser that would normally have a fixed numerical aperture determined by the geometry and proximity of the reflecting surfaces. For the purposes of this tutorial, we have modified the light path through the condenser to mimic changes in numerical aperture that do not correlate with the actual shape of the condenser.

Specimen Illumination With Darkfield Condensers - This tutorial explores how a darkfield microscopy specimen scatters light into the objective when illuminated with a hollow cone of oblique illumination from a Cardioid darkfield condenser. As the sample encounters light from the Cardioid condenser, it refracts, diffracts, and scatters the light into the objective. The physical concepts behind this tutorial do not rely specifically on the Cardioid-type condenser, and the same principles apply to all darkfield condensers.

Catadioptric Darkfield Reflected Light Objectives - Discover how light is scattered and diffracted by a specimen into the front lens of a darkfield reflected light objective. Light enters the hollow periphery of the objective (which serves as a condenser in this situation) and is reflected through a series of lenses and mirrored surfaces. The focused light is then projected into an apex that illuminates the specimen at an oblique angle from every azimuth.

Phase Contrast Microscopy

Phase Plate/Ring Alignment - Concentric alignment of the condenser phase plate slits with the phase ring, positioned inside the objective, is of paramount importance in phase contrast microscopy. This tutorial explores the effect of phase plate/ring alignment on specimen contrast using this important microscopy technique.

Apodized Phase Contrast - In apodized phase contrast microscopy, halo attenuation and an increase in specimen contrast can be obtained by the utilization of selective amplitude filters located adjacent to the phase film in the phase plates built into the objective at the rear focal plane. These amplitude filters consist of neutral density filter thin films applied to the phase plate surrounding the phase film as demonstrated in this interactive tutorial.

Specimen Contrast Enhancement with Apodized Phase Plates - The purpose of phase rings in microscope objectives is to retard the phase of direct light passing through the specimen by one quarter wavelength to allow constructive and destructive interference with diffracted light at the intermediate image plane. Examine how neutral density material surrounding phase rings can act to improve contrast by reducing the halo effect.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Kirill I. Tchourioukanov and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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