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Optical Microscopy

1. Resolution

2. Visability

3. Special Subjects

4. Glossary

5. References

b
   
Optical Microscopy

This paper was presented for translators at the 37th Annual Conference of the American Translators Association, 1996.
Copyright: American Translators Association. All rights reserved. This document may not be reproduced without the written permission of the American Translators Association. Presented here by permission, and slightly modified for this web page.

OPTICAL MICROSCOPY

Denzel L. Dyer
Dyer Scientific and Technical Translations


Abstract: Optical microscopy attempts to make small clear colorless objects visible and to show fine detail in them. This paper gives a survey of the most important such attempts.

The optical microscope is one of the very oldest scientific instruments. It has been developed essentially up to its theoretical limits. It appears to be simple and easy to understand and use, but that appearance is deceiving. Probably most people who use microscopes do not know how to use them most effectively, and they may use terms incorrectly.

Most people think of microscopes just as instruments which magnify. They do magnify, of course, but magnification alone is relatively easy and almost always inadequate. The real objective of using a microscope is usually to see fine detail in the object being studied. As most of the objects examined are either colorless and transparent, or opaque, most progress in microscopy has involved ways to increase resolution (ability to distinguish fine structure) or ways to make invisible objects visible.

1.     RESOLUTION

Ability to distinguish fine structure is increased by correcting lens aberrations and by increasing numerical aperture. The principal lens aberrations are chromatic and spherical aberration. Corrections require rather complex lens systems with several elements made of different materials.

With a single lens (one element, one material), the image is focused because the lens has a refractive index greater than that of the surrounding air. The refractive index varies with the wavelength of the light (dispersion), so that the image from a simple lens always has colored fringes (chromatic aberration). Optical engineers have produced various multielement systems to correct chromatic aberration. Essentially all microscope optical systems are now at least 'achromatic': corrected so that two colors of light focus at the same plane. This still has some color fringing, but is often quite acceptable. Many systems are 'apochromatic': corrected so that three or even four colors focus at the same plane. These are more expensive but distinctly better for color photomicrography. In some apochromatic systems, the correction is divided between the objective and the ocular lens, so that a special 'compensating' ocular must be used. Some objectives without completely apochromatic correction are 'semiapochromats'. Microscope objectives sometimes contain a lens element made of fluorspar (CaF2), and are called 'fluorites'. Until recently, other materials were limited to 'crown' glass and 'flint' glass, but now optical glasses are also made from rare earth elements, and corrections are improved. The entire problem of color correction can be avoided by using monochromatic illumination. That is sometimes done in microscopy, and is usual in 'microphotography' (making very small images, as of microcircuits). Resolution is better at short wavelengths, so some systems are designed to form images with ultraviolet light.

With a single lens in which the surfaces are slices of spheres, light rays passing through the center of the lens are focused at a different position than those passing through the outer parts of the lens (spherical aberration). A lens can be corrected for spherical aberration by using multiple elements. Such a lens is called 'aplanatic'. This is usually assumed for microscope objectives, but often mentioned for condensers. Spherical aberration can also be reduced by grinding lenses with one or more surfaces that are not spherical; these lenses are 'aspheric'.

No matter how well corrected the lens system is, all lens systems produce images in which the edges are slightly blurred. A very find structure appears only as a blur. Measurements are uncertain because of the blur, and two edges which are very close together may not be distinguished. The ability of the system to show points or edges clearly is expressed by its 'resolution' or 'resolving power'. In this context, resolving power is the distance by which two points must actually be separated in order for an observer to see that they are separate. No matter how well corrected the lens may be, its resolving power is limited by its numerical aperture (NA) and by the wavelength of light used (l). The relation usually accepted is:

                                Resolving power = 0.6 l/NA

                                          NA = n sin u

where n is the lowest refractive index of the various media in the optical path between the condenser and the objective lens; l is the wavelength of the light used; and u is the angle between the optical axis and the most divergent ray that can still enter the objective lens. The angle u (half the 'angular aperture') is limited to about 75 (sin 75 = 0.966). If the light passes through air between the condenser and objective, n is 1.00, and the maximum numerical aperture is about 0.95. With immersion systems in which the light passes only through immersion oil or glass, n can be as high as 1.65 (usually 1.515) and the numerical aperture could be as high as 1.5 (usually not more than 1.4). Although many users do not realize it, the illuminating system (condenser) must have approximately the same numerical aperture as the objective lens. The numerical aperture of the system is often considered to be the mean of the condenser and objective numerical apertures.

Because the resolving power is limited, increasing magnification arrives at the point where higher magnification makes the image bigger but does not show any more detail.

Useful magnification (visual observation) = 500 to 1000 (NA)
(Higher for a projected image or print to be viewed from a distance)
Magnification higher than 'useful' is called 'empty'. Very often, the maximum useful magnification is not needed. Lenses of lower magnification and lower numerical aperture which do not require immersion are much less expensive, much easier to use (longer working distance), and have much larger fields of view and depth of field.

As noted above, the illumination system itself is important: it should have a numerical aperture about as high as that of the objective lens, and should illuminate the object evenly. For a transparent object, the illuminating light usually passes through the object and into the objective lens (transmitted light). For an opaque object the light must be directed onto the side of the object toward the objective lens. This is 'incident light', sometimes called 'top light' or 'epi-illumination'. Sometimes, with either incident or transmitted illumination, the light comes from just one side. This is 'oblique' illumination.

Illumination must be relatively intense. All the light for the image must come from the object, and when an object is magnified by a factor of 100 the light on the object is spread over an image area 10,000 times greater, so the intensity is lower by the same factor.


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