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The Optical Qualities of Microscope Objectives. Resolving power, depth of sharpness, illuminating power, chromatic aberration, spherical aberration, flatness of field and working distance for lenses.
Optical Qualities of Microscope Objectives
Numerical Aperture. N. A.=n. sin u. This term was introduced by Abbe. N. is the refractive index of the medium between the object and the front lens of the objective (air in case of dry objectives and water or oil in case of immerson objectives), and u is half the angular aperture. Important Qualities for Objectives Several important qualities of the objective depend upon the numerical aperture. (a) RESOLVING POWER. This is directly proportional to the numerical aperture and represents the ability of the objective to show detail in the image of the object. The higher the numerical aperture, the greater the resolving power, and the finer the detail we may expect to see in the image. (b) DEPTH OF SHARPNESS, OR PENETRATION. This is the power of an objective to show sharply objects lying in different planes, one above another, without the necessity of focusing up and down. The depth of sharpness is in inverse ratio to the numerical aperture. Therefore the lens of low numerical aperture has little resolving power and great penetration. The lens of high numerical aperture has great resolving power and little penetration, unless it be used with a narrow cone of light which practically makes it a lens of low aperture with the qualities of such a lens. (c) ILLUMINATING POWER. The brilliancy of the objective increases with the square of the numerical aperture of the objective. An objective of .40 N. A. will give an image four times as brilliant as one of .20 N. A., provided the magnification is the same and the full cone of the illumination is used in both cases. Magnifying Power. The magnifying power of an objective is in inverse ratio to its focal distance. An objective of 2mm focal distance will give, with the same ocular, a magnification eight times greater than one of 1 6mm focal distance. Numerical aperture and magnifying power are of little advantage if the definition is not good. Definition. The definition of an objective is characterized by the cleanness and sharpness of the outlines of the image. Definition depends upon the corrections for chromatic and spherical aberrations, and the workmanship; the centering of the lenses, etc. CHROMATIC ABERRATION is due to the fact that a ray of white light passing from one medium to another of different refractive index at any angle other than 90 to the surface between them is refracted and dispersed into its component colors. SPHERICAL ABERRATION is due to the fact that a spherical surface cannot bring a beam of light which passes through its vertex to the same focus as that of a beam of light passing through any other zone. Both aberrations are corrected by the use of different kinds of glass (crown and flint) combined as double and triple lenses in the objective. Neither can be corrected absolutely for all colors in an achromatic objective. Apochromatic objectives approach the ideally corrected objective almost to perfection. An objective can be tested for chromatic correction by using a narrow cone of oblique light and a coarse grating. Abbe's test plate is best. Diatoms are good. No stained object should be used. If the spherical correction is perfect (see next paragraph) and one side of a line passing through the center of the field shows a clear, narrow, greenish yellow border, while the other side is fringed with a violet red (seconddary colors) the objective is chromatically corrected. The colors shown in the higher power objectives are of a more primary character, /. E., nearer the yellow and blue. Apochromatic objectives showno color borders in this test. The spherical correction of an objective is perfected for a certain thickness of cover glass and a certain tube length, and is influenced greatly by any variation in either. This is especially true with the high power dry objectives. The homogeneous immersion objectives are not sensitive to the variation in the cover thickness because the immersion oil between the cover glass and the lens is of the same refractive index as the glass. They must be used however, with the proper tube length. In testing an objective for its spherical correction it is therefore very important to supply the proper thickness of cover and tube length. It is manifestly unfair to judge an objective on this point without complying with these conditions. The test for spherical correction can be made on the same object as used for the chromatic test. If the edges of the lines in the center of the field appear equally sharp and clear when illuminated by either a narrow central cone of light or a narrow oblique cone without having to change the fine adjustment the objective is spherically corrected. The color remnants mentioned above will be clear and transparent, while, if the lens is poorly corrected spherically, these borders will appear muddy and turbid. Defects in spherical corrections can often be corrected by using cover glasses suitable to them, also by changing the tube length. The fact that the periphery of the field is not in focus at the same time as the center does not bespeak a lack of spherical correction, but a lack of flatness of field with which it is often confounded. Flatness of Field depends not only upon the objective itself, but upon the ocular and the cone of light used, whereas the spherical aberration is inherent in the objective itself. No field is absolutely flat. It is a desirable quality in a lens but spherical and chromatic corrections should never be sacrificed for it. Some lenses appear to be "flatter" than they really are, because their corrections are so poor that little contrast is noticed between objects in the center of the field and at the edge. Narrow cones of light give a flatter field than wide ones. Thin objects are more critical tests for flatness of field than thick ones. Working Distance is the free distance between the cover glass and the objective when the latter is foccrsed. It decreases generally with increasing power and numerical aperture of the objective. Of two lenses with the same focal distance the one with the higher N. A. will have the shorter working distance. The working distance also depends on the mounting of the front lens. If the lens has a prominent mounting projecting beyond its surface the working distance is lessened thereby. The Oculars A certain magnification by the ocular will be necessary, and sufficient, to bring out all the detail in the image which can be secured from the numerical aperture of the objective. If we use a higher ocular we lose depth of sharpness and size of field, since they are both inversely proportional to the magnification. We also lose illumination, which varies inversely as the square of the magnification. We therefore get the greatest effectiveness out of an objective, the largest field, the greatest penetration, and the best illumination, by using the lowest magnification which makes all the detail in the image visible. If we increase the magnification beyond this point we do so at the expense of other good qualities. Lengthening the tube increases the magnification proportionately.
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Microscope Equipment 
