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Specialized Microscopy Techniques Interactive Tutorials

Section Overview:

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 by plotting image contrast as a function of image intensity and displaying the contrast range for each background intensity selected by the slider.

  • Optical Gradients in Phase Objects

    See how light passes through a phase object whose sides are not plano-parallel but rather appear as a prism as light passes through the phase object from the bottom and travels through, exiting at the top on the rounded surface.

  • Optical Path Difference

    Explore optical path differences for phase objects as a function of specimen and surrounding medium refractive index variations. Optical path difference is the product of two terms: thickness (t) and difference in refractive index (n).

  • 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.

  • Reflected Light Microscopy

    Examine the optical pathways in a reflected light microscope in this interactive tutorial. The visitor can open and close the iris diaphragms controlling the condenser and the field lens.

  • Rheinberg Illumination

    Discover the changes in specimen appearance when the colors are varied in the annular and central filters used in Rheinberg illumination in this featured interactive java tutorial.

  • Spherulites in Polarized Light

    Explore how crystals with spherulitic growth patterns appear under a polarized light microscope in this interactive java tutorial. As the applet initializes, the sample will slowly spin.

  • Cardioid Condensers

    Explore how a darkfield microscopy specimen scatters light into the objective when illuminated with a hollow cone of oblique illumination from a Cardioid darkfield condenser in this interactive tutorial.

Hoffman Modulation

  • Modulation Contrast

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

  • Optical Sectioning

    Explore how focus can be varied on thick samples to selectively bring different focal planes into view. Optical sectioning is a technique that is possible with techniques such as Hoffman modulation contrast.

  • Hoffman Modulation Contrast

    Examine how rotation of the substage polarizing filter affects image contrast in Hoffman modulation contrast microscopy. Instructions for operation of the tutorial appear below the applet window.

  • Hoffman Modulation Contrast: Slit Alignment

    Explore how the slit is imaged and aligned with respect to the modulator in Hoffman modulation contrast microscopy. Instructions for use of the tutorial are listed below the applet window.

Darkfield Microscopy

  • Darkfield Condenser Adjustment

    Visitors and students are allowed to experiment with the adjustable parameters in condenser alignment and configuration for darkfield microscopy in this interactive java tutorial.

  • Hollow Light Cone Numerical Aperture

    Examine how the shape and size of the hollow cone of light emitted by a hypothetical reflecting darkfield condenser changes with numerical aperture in this interactive java tutorial.

  • Light Cone Formation with Abbe Darkfield Condensers

    Explore how a changes in the size of the opaque stop in a simple Abbe darkfield illumination condenser affects light cone shape and numerical aperture in this tutorial.

  • Catadioptric Darkfield Reflected Light Objectives

    Discover how light that is diffracted, refracted, and reflected by a specimen enters the front lens of a darkfield objective in reflected light microscopy.

Phase Contrast Microscopy

  • Phase Plate/Ring Alignment

    Students can learn and explore the effect of phase plate/ring alignment on specimen contrast using this important microscopy technique in this interactive java tutorial.

Differential Interference Contrast

  • Optical Path Gradients and Intensity Profiles

    Explore the relationship between optical path gradients and amplitude (intensity) profiles for a variety of semi-transparent specimens in this featured interactive java tutorial.

  • The Interference Background Image

    Examine how changes to the optical path difference between orthogonal wavefronts can produce a range of interference colors that are useful in determining path lengths in this interactive tutorial.

  • Optical Staining with DIC Microscopy

    Discover and learn how varying the amount of bias retardation can affect the appearance and level of staining achieved in the specimen image in this interactive java tutorial.

  • de Sénarmont Compensators

    A de Sénarmont compensator is composed of a polarizer combined with a quarter-wavelength retardation plate. See the relationship between wavefronts emanating from the compensator as the polarizer is rotated through its range.

  • Optical Sectioning with Phase Contrast & DIC

    Compare optical sectioning of thick specimens with DIC and phase contrast, as it reveals the benefits of unrestricted aperture effects on obtaining well-defined sections in this interactive tutorial.

  • Wavefront Shear in Wollaston and Nomarski Prisms

    Explore differences between the location of the interference plane in both prism types, and how the position of the plane can be varied with changes to the optical axis orientation.

  • Differential Interference Contrast (DIC)

    Explore how changes in the Wollaston prism position affects how images are seen in the microscope viewfield. The tutorial permits addition of a retardation plate to the optical pathway to add color and contrast to the specimen.

Oblique Illumination

  • Oblique Illumination Light Pathways

    Discover changes in microscope light paths and demonstrates events at the objective rear focal plane as illumination progresses from axial to highly oblique in this interactive java tutorial.

  • Oblique Illumination Refractive Index Determination

    Explore how variations in the refractive index of a specimen and its surrounding medium alter visibility in the microscope when utilizing oblique illumination techniques.

Near-Field Scanning Optical Microscopy

  • Aperture Scanning of a Line Grating

    Learn more about E. A. Ash and G. Nicholls demonstration of the near-field resolution of a subwavelength aperture scanning microscope operating in the microwave region of the electromagnetic spectrum in this tutorial.

  • Van der Waals Forces

    In the near-field scanning microscopy configuration, several forces exist between the probe tip and the specimen. Examine the dependence of these forces on the distance between the NSOM probe tip and the specimen.

  • Unique NSOM Reflection Mode

    A unique (and more difficult) configuration for NSOM imaging of opaque specimens, which can produce exceptional results is presented in this interactive java tutorial.

  • Near-Field Scanning Optical Microscopy Simulation

    Explore the difference between scanning with the probe in feedback mode, which the tip height varies in response to specimen topography, and scanning without feedback.

  • Mechanical Oscillator

    The mechanical system examined in this tutorial represents the interaction of these parameters for both the tuning fork oscillator and the bent optical probe NSOM configurations.

  • NSOM Probe Aperture Throughput

    Learn about NSOM Probe Aperture Throughput as both the wavelength of the light passing through the aperture and the aperture diameter can be manipulated by adjusting the value of the Wavelength and Aperture Diameter slider bars.

  • Thermal Effects on NSOM Probes

    Learn more about the effects of thermal heating of the NSOM probe, which occurs in the taper region due to the absorption of light by the metallic coating engulfing the exterior of the probe.

Fluorescence Microscopy

  • 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.

  • Fluorescence Filter Cubes

    Explore how excitation and barrier filters can be interchanged to permit a wide spectrum of specific wavelengths to probe fluorescence samples in this interactive fluorescence tutorial.

  • 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.

  • Jablonski Energy Diagram

    Explore how electrons in fluorophores are excited from the ground state into higher energy states, and the events that occur as these molecules relax by photon emissionto ultimately fall back into the ground-level energy state.

  • Photobleaching

    Photobleaching occurs when a fluorophore permanently loses the ability to fluoresce. Explore variations in photobleaching rates in single, dual, and multiply labeled fluorescence specimens in this tutorial.

  • Solvent Effects on Fluorescence Emission

    The high degree of sensitivity in fluorescence is due to interactions that occur in the local environment during the excited state. Examine relaxation effects and associated spectral shifts that occur as a function of solvent polarity.

  • Olympus BX51 Microscope Lightpath

    Explore illumination pathways in the Olympus BX51 research-level upright microscope. The microscope drawing presented in the tutorial illustrates a cut-away diagram of the Olympus BX51 microscope.

  • Olympus IX70 Microscope Lightpath

    Explore light pathways through an inverted tissue culture microscope equipped with for both diascopic (tungsten-halogen) and epi-fluorescence (mercury arc) illumination in this interactive tutorial.

  • Interference Filters

    Explore how interference filters operate by selectively transmitting constructively reinforced wavelengths while simultaneously eliminating unwanted light with this interactive tutorial.

  • Liquid Crystal Tunable Filters

    The LCTF (Liquid Crystal Tunable Filters) utilized in this tutorial is an example of a Lyot filter, in which four polarizers are separated by three layers of liquid crystals sandwiched between birefringent crystals.

  • Matching Filter Blocks with Probes

    Examine the fluorophore characteristics necessary to maximize the efficiency of excitation and emission in conjunction with current Olympus fluorescence filter blocks in this interactive tutorial.

  • Acousto-Optic Tunable Filters

    The acousto-optic tunable filters (AOTF) are increasingly being employed to modulate the wavelength and amplitude of illuminating laser light in the latest generation of confocal microscopes as shown in this tutorial.

  • Multiphoton Jablonski Diagram

    Two-photon and three-photon excitation occurs as the result of simultaneous fluorophore absorption by photons in a single quantitized event. Explore how fluorescence excitation events occur in multiphoton microscopy.

  • Excitation Region Events

    Examine events occurring in the microscope focal region during specimen excitation using long wavelength visible and near infrared laser illumination in this interactive java tutorial.

  • Reflected Light (Episcopic) Fluorescence Microscopy

    Discover and learn more about the light pathways in a reflected light (episcopic) fluorescence microscope using this featured interactive java tutiorial featuring a cut-away diagram.

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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