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Interactive Java Tutorials

We have constructed a variety of Java tutorials designed to help students grasp the fundamental details of basic microscopy principals. Please use the links below to visit the tutorials in our collection.

Simple Magnification - Explore how a simple magnifying glass works with this tutorial designed to explain the concept of magnification. The visitor can use the mouse cursor to move a magnifying glass to change the image enlargement of an object.

Transmitted Light Microscopy - This tutorial explores the optical pathways in a typical transmitted light microscope. The visitor is able to control both the field and condenser iris diaphragms to vary the amount of light admitted into the microscope.

Numerical Aperture - A tutorial designed to illustrate how the angular aperture of a microscope objective is related to the refractive index of a medium and how the numerical aperture varies with the value of the angular aperture.

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

Substage Condenser Numerical Aperture - A tutorial that explains how condenser numerical aperture should be adjusted to coincide with objective magnification. Explore how the condenser light cone is changed with changes in numerical aperture.

The Condenser Aperture Diaphragm - Explore how incorrect use of the substage condenser aperture diaphragm can cause excessive glare and washing out of the sample and also how closing it too much will result in increased diffraction and loss of resolution.

Condenser Light Cones - Substage condensers produce unique light cones dependent partially upon the degree of optical correction. This tutorial explores the various light cones produced by condensers of increasing optical correction.

The Field Diaphragm - This aperture iris diaphragm controls how much light enters the microscope. The tutorial allows the visitor to open and close the diaphragm and to see how this affects an image in the viewfield.

Immersion Oil - The numerical aperture of an objective is partially dependent upon the refractive index of the imaging medium. This tutorial explores how changes in this value affect the light cone entering the objective.

Objective Focal Length - The focal length of an objective is dependent upon the magnification and the microscope tube length. This tutorial examines how changes in the tube length affect focal length in infinity-corrected microscopes.

Alignment of the Lamp Filament - Alignment of the microscope illuminator lamp filament is of fundamental importance in achieving Köhler illumination. This tutorial allows the student to experiment with the focus and alignment of the microscope lamp filament.

Lasers - This tutorial explores how a ruby laser crystal works when excited by a xenon flash tube. Lasers are sometimes useful as light sources in optical microscopy.

Microscope Optical Train Java Tutorials

Geometrical Construction of Ray Diagrams - A popular method of representing a train of propagating light waves involves the application of geometrical optics to determine the size and location of images formed by a lens or multi-lens system. This tutorial explores how two representative light rays can establish the parameters of an imaging scenario.

Perfect Lens Characteristics - The simplest imaging element in an optical microscope is a perfect lens, which is an ideally corrected glass element that is free of aberration and focuses light onto a single point. This tutorial explores how light waves propagate through and are focused by a perfect lens.

Perfect Two-Lens System Characteristics - During investigations of a point source of light that does not lie in the focal plane of a lens, it is often convenient to represent a perfect lens as a system composed of two individual lens elements. This tutorial explores off-axis oblique light rays passing through such a system.

Projection and Viewing Eyepieces - The eyepiece (or ocular) is designed to project either a real or virtual image, depending upon the relationship between the intermediate image plane and the internal eyepiece field diaphragm. Explore how eyepieces can be coupled to the human eye or a camera system to produce images generated by the microscope objective.

Condenser Image Planes - In a microscope optical system, the lamp filament is imaged in the focal plane of the condenser aperture diaphragm when the microscope is configured to operate under conditions of Köhler illumination. This tutorial explores the relationship between image planes relevant to the field and condenser diaphragms and how aperture size affects ray trace pathways.

Microscope Conjugate Field Planes - In a microscope optical system, the lamp filament is imaged in the focal plane of the condenser aperture diaphragm when the microscope is configured to operate under conditions of Köhler illumination. This tutorial explores the relationship between image planes relevant to the field and condenser diaphragms and how aperture size affects ray trace pathways.

Infinity Microscope Conjugate Field Planes - The geometrical relationship between image planes in the optical microscope configured for infinity correction with a tube lens is explored in this tutorial. In such a microscope, magnification of the intermediate image is determined by the ratio of the focal lengths of the tube lens and objective lens.

Image Formation Java Tutorials

Airy Pattern Formation - When an image is formed in the focused image plane of an optical microscope, every point in the specimen is represented by an Airy diffraction pattern having a finite spread. This occurs because light waves emitted from a point source are not focused into an infinitely small point by the objective, but converge together and interfere near the intermediate image plane to produce a three-dimensional Fraunhofer diffraction pattern.

Airy Pattern Basics - When the diffraction pattern formed by a specimen in the microscope is sectioned in the focal plane, it is observed as the classical two-dimensional diffraction spectrum known as the Airy pattern. This tutorial explores how Airy pattern size changes with objective numerical aperture and the wavelength of illumination; it also simulates the close approach of two Airy patterns.

Light Diffraction Through a Periodic Grating - A model for the diffraction of visible light through a periodic grating is an excellent tool with which to address both the theoretical and practical aspects of image formation in optical microscopy. Light passing through the grating is diffracted according to the wavelength of the incident light beam and the periodicity of the line grating. This interactive tutorial explores the mechanics of periodic diffraction gratings when utilized to interpret the Abbe theory of image formation in the optical microscope.

Numerical Aperture and Image Resolution - The image formed by a perfect, aberration-free objective lens at the intermediate image plane of a microscope is a diffraction pattern produced by spherical waves exiting the rear aperture and converging on the focal point. This tutorial explores the effects of objective numerical aperture on the resolution of the central bright disks present in the diffraction pattern, commonly known as Airy disks.

Conoscopic Images of Periodic Gratings - The purpose of this tutorial is to explore the reciprocal relationship between line spacings in a periodic grid (simulating a specimen) and the separation of the conoscopic image at the objective aperture plane. When the line grating has broad periodic spacings, several images of the condenser iris aperture appear in the objective rear focal plane. If white light is used to illuminate the line grating, higher order diffracted images of the aperture appear with a blue fringe closer to the zeroth order (central) image and with a green-yellow-red spectrum appearing further out towards the objective aperture periphery.

Spatial Frequency and Image Resolution - When a line grating is imaged in the microscope, a series of conoscopic images representing the condenser iris opening can be seen at the objective rear focal plane. This tutorial explores the relationship between the distance separating these iris opening images and the periodic spacing (spatial frequency) of lines in the grating.

Airy Patterns and the Rayleigh Criterion - Airy diffraction pattern sizes and their corresponding radial intensity distribution functions are sensitive to both objective numerical aperture and the wavelength of illuminating light. For a well-corrected objective with a uniform circular aperture, two adjacent points are just resolved when the centers of their Airy patterns are separated by a distance r. This tutorial examines how Airy disk sizes, at the limit of optical resolution, vary with changes in objective numerical aperture and illumination wavelength and how these changes affect the resolution of the objective.

Axial Resolution and Depth of Field - The lateral resolution for an Airy diffraction pattern generated by a point light source is defined within a single plane of focus at the intermediate image position in an optical microscope. In fact, the diffraction image of a point source extends periodically and symmetrically above and below this plane into a three-dimensional pattern that expands and spreads out from the center along the optical axis. This tutorial explores the structure of cross sections taken along the optical axis of the microscope near the focal plane using a virtual high numerical aperture objective free from spherical aberration.

Periodic Diffraction Images - When a microscope objective forms a diffraction-limited image of an object, it produces a three-dimensional diffraction pattern that is periodic both along the optical axis and laterally within the intermediate image plane. This tutorial explores diffraction images produced by a periodic object at several focal depths.

Optical Aberration Java Tutorials

Astigmatism - Astigmatism aberrations are similar to comatic aberrations, however these artifacts are not as sensitive to aperture size and depend more strongly on the oblique angle of the light beam. The aberration is manifested by the off-axis image of a specimen point appearing as a line or ellipse instead of a point. Depending on the angle of the off-axis rays entering the lens, the line image may be oriented in either of two different directions, tangentially (meridionally) or sagittally (equatorially). The intensity ratio of the unit image will diminish, with definition, detail, and contrast being lost as the distance from the center is increased.

Chromatic Aberration - Chromatic aberrations are wavelength-dependent artifacts that occur because the refractive index of every optical glass formulation varies with wavelength. When white light passes through a simple or complex lens system, the component wavelengths are refracted according to their frequency. In most glasses, the refractive index is greater for shorter (blue) wavelengths and changes at a more rapid rate as the wavelength is decreased.

Comatic Aberration - Comatic aberrations are similar to spherical aberrations, but they are mainly encountered with off-axis light fluxes and are most severe when the microscope is out of alignment. When these aberrations occur, the image of a point is focused at sequentially differing heights producing a series of asymmetrical spot shapes of increasing size that result in a comet-like (hence, the term coma) shape to the Airy pattern.

Curvature of Field - Modern microscopes deal with field curvature by correcting this aberration using specially designed objectives. These specially-corrected objectives have been named plan or plano (for flat-field) and are the most common type of objective in use today, providing ocular fields ranging between 18 and 26 millimeters, which exhibit sharp detail from center to edge.

Geometrical Distortion - Distortion is an aberration commonly seen in stereoscopic microscopy, which is manifested by changes in the shape of an image rather than the sharpness or color spectrum. The two most prevalent types of distortion, positive and negative (often termed pincushion and barrel, respectively), can often be present in very sharp images that are otherwise corrected for spherical, chromatic, comatic, and astigmatic aberrations. In this case, the true geometry of an object is no longer maintained in the image.

Spherical Aberration - The most serious of the monochromatic defects that occurs with microscope objectives, spherical aberration, causes the specimen image to appear hazy or blurred and slightly out of focus. The effect of spherical aberration manifests itself in two ways: the center remains more in focus than the edges of the image and the intensity of the edges falls relative to that of the center. This defect appears in both on-axis and off-axis image points.

Focus Depth and Spherical Aberration - The lateral resolution for an Airy diffraction pattern generated by a point light source is defined within a single plane of focus at the intermediate image position in an optical microscope. When the aperture function of an objective is non-uniform, or in the case of spherical aberration, the wavefront leaving the lens is no longer spherical with a center positioned at the point of focus in the image plane. Instead, the wavefront is distorted and departs from ideal behavior in a manner that is dependent upon the nature of the aberration and/or image filters and conditions that are present in the optical system. At the intermediate image plane, the point spread function yields an asymmetrical distribution where the intensity ratio between the central peak and surrounding rings is shifted with the latter becoming far more prominent.

Cover Glass Thickness Correction - High magnification objectives designed to be used with air as the immersion medium between the front lens and the cover glass are prone to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for these fluctuations.

Contributing Authors

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

H. Ernst Keller - Carl Zeiss Inc., One Zeiss Dr., Thornwood, NY, 10594.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Brian O. Flynn, John C. Long, Matthew J. Parry-Hill, 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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