An ideal microscope objective produces a symmetrical diffraction limited image of an Airy pattern from an infinitely small object point. The image plane is generally located at a fixed distance from the objective front lens in a medium of defined refractive index. Microscope objectives offered by the leading manufacturers have remarkably low degrees of aberrations and other imperfections, provided the appropriate objective is selected for the task and the objective is utilized properly in accordance with the manufacturer's recommendations. It should be emphasized that objective lenses are not made to be perfect from every standpoint, but are designed to meet certain specifications depending on their intended use, constraints on physical dimensions, and price ranges.
Objectives are made with differing degrees of optical correction for both monochromatic (spherical, astigmatism, coma, distortion) and polychromatic aberrations, field size and flatness, transmission wavelengths, freedom from fluorescence, birefringence and other factors contributing to background noise. Depending upon the degree of correction, objectives are generally classified as achromats, fluorites, and apochromats, with a plan designation added to lenses with low curvature of field and distortion. This section addresses some of the more common optical aberrations that are commonly found (and often corrected) in microscope objectives.
Overview of Optical Aberrations - Departures in lens action from the idealized conditions of Gaussian optics are known as optical aberrations. Microscope optical trains typically suffered from as many as five common aberrations: spherical, chromatic, curvature of field, comatic, and astigmatic. Geometrical distortion is another artifact often encountered in eyepieces and the zoom lens systems found in stereoscopic microscopes.
Field Curvature - A simple lens focuses image points from an extended flat object, such as a specimen on a microscope slide, onto a spherical surface resembling a curved bowl. The nominal curvature of this surface is the reciprocal of the lens radius and is referred to as the Petzval Curvature of the lens. Curvature of field in optical microscopy is an aberration that is familiar to most experienced microscopists.
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.
Adjustment of Objective Correction Collars - Most microscope objectives are designed to be used with a cover glass that has a standard thickness of 0.17 millimeters and a refractive index of 1.515, which is satisfactory when the objective numerical aperture is 0.4 or less. However, when using high numerical aperture dry objectives (numerical aperture of 0.8 or greater), cover glass thickness variations of only a few micrometers result in dramatic image degradation due to aberration, which grows worse with increasing cover glass thickness. To compensate for this error, the more highly corrected objectives are equipped with a correction collar to allow adjustment of the central lens group position to coincide with fluctuations in cover glass thickness. This interactive tutorial explores how a correction collar is adjusted to achieve maximum image quality.
Selected Literature References - Aberrations are divided into two main categories: errors that occur when polychromatic light (white light) is passed through a lens, and errors that are present when only a single wavelength (monochromatic) of light is utilized. The selected references listed in this section contain information about the cause and correction of the most common optical aberrations encountered with microscope and other lens systems. Bear in mind that the optical designer must correct for both polychromatic and monochromatic aberrations simultaneously in the production of well-corrected microscope objectives.
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, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
Questions or comments? Send us an email.
© 1998-2021 by Michael W. Davidson and The Florida State University. All Rights Reserved. No images, graphics, scripts, or applets may be reproduced or used in any manner without permission from the copyright holders. Use of this website means you agree to all of the Legal Terms and Conditions set forth by the owners.
This website is maintained by our