Explain why parfocal imaging is important

The objective is parfocalized by translating the entire lens cluster upward or downward within the sleeve with locking nuts so that objectives housed on a multiple nosepiece can be interchanged without losing focus. Adjustment for coma is accomplished with three centering screws that can optimize the position of internal lens groups with respect to the optical axis of the objective. There is a wealth of information inscribed on the objective barrel. Briefly, each objective has inscribed on it the magnification e.

If the objective is designed to operate with a drop of oil between it and the specimen, the objective will be engraved OIL or OEL or HI homogeneous immersion. In cases where these latter designations are not engraved on the objective, the objective is meant to be used dry, with air between the lowest part of the objective and the specimen.

Objectives also always carry the engraving for the numerical aperture NA value. This may vary from 0. If the objective carries no designation of higher correction, one can usually assume it is an achromatic objective. More highly corrected objectives have inscriptions such as apochromat or apo, plan, FL, fluor, etc.

Older objectives often have the focal length lens-to-image distance engraved on the barrel, which is a measure of the magnification. In modern microscopes, the objective is designed for a particular optical tube length, so including both the focal length and magnification on the barrel becomes somewhat redundant.

Table 1 lists working distance and numerical aperture as a function of magnification for the four most common classes of objectives: achromats , plan achromats , plan fluorites , and plan apochromats. Note that dry objectives all have a numerical aperture value of less than 1. When a manufacturer's set of matched objectives, e. Thus, changing objectives by rotating the nosepiece usually requires only minimal use of the fine adjustment knob to re-establish sharp focus.

Such a set of objectives is described as being parfocal, a useful convenience and safety feature. Matched sets of objectives are also designed to be parcentric, so that a specimen centered in the field of view for one objective remains centered when the nosepiece is rotated to bring another objective into use. For many years, objective lenses designed for biological applications from most manufacturers all conformed to an international standard of parfocal distance.

Thus, a majority of objectives had a parfocal distance of With the migration to infinity-corrected tube lengths, a new set of design criteria emerged to correct for aberrations in the objective and tube lenses. Coupled to an increased demand for greater flexibility to accommodate the need for ever-greater working distances with higher numerical apertures and field sizes, interchangeability between objective lenses from different manufacturers disappeared.

This transition is exemplified by the modern Nikon CFI optical system that features "Chrome Free" objectives, tube lenses, and eyepieces. Each component in the CFI system is separately corrected without one being utilized to achieve correction for another.

The tube length is set to infinity parallel light path using a tube lens, and the parfocal distance has been increased to 60 millimeters.

Even the objective mounting thread size has been altered from The field diameter in an optical microscope is expressed by the field-of-view number or simply field number , which is the diameter of the viewfield expressed in millimeters and measured at the intermediate image plane. The field diameter in the object specimen plane becomes the field number divided by the magnification of the objective.

Begin by trying to turn the ring. If the ring moves then you may skip the next image and set of instructions. If the ring does not move it is most likely because there is a dab or drop of optical cement that is keeping it from rotating. You may have to look closely in order to locate the drop of optical cement holding the ring in place.

You can remove the optical cement on the objective adjustment ring. If it can't be removed with your finger nail you may want to lightly moisten a Q-tip with acetone and carefully remove the dab of cement. You may need to work the ring back and forth to make the ring spin freely. After the ring is free re-install the objective this time without the cover on it on the microscope.

Make sure you put the next highest objective after the 10x so the objectives ascend in order. For example, place the 10x, then 40x, x, etc. Rotate the 10x objective into position and focus on an object. A stage micrometer is a good item to focus on, but you can use anything with lines on it. Now move up to the next objective. An even higher level of correction and cost is found in objectives called fluorites or semi-apochromats illustrated by center objective in Figure 2 , named for the mineral fluorite, which was originally used in their construction.

Fluorite objectives are fashioned from advanced glass formulations that contain materials such as fluorspar or newer synthetic substitutes that allow for greatly improved correction of optical aberration. Similar to the achromats, the fluorite objectives are also corrected chromatically for red and blue light, however, the fluorites are also spherically corrected for two or three colors instead of a single color, as are achromats.

Compared to achromats, fluorite objectives are made with a higher numerical aperture, which results in brighter images. Fluorite objectives also have better resolving power than achromats and provide a higher degree of contrast, making them better suited for color photomicrography in white light. The third type of objective, the apochromatic objective, possesses the highest level of correction Figure 2. Lower power apochromat objectives 5x, 10x, and 20x have a longer working distance than higher power 40x and x apochromat objectives.

Apochromats almost eliminate chromatic aberration, are usually corrected chromatically for three colors red, green, and blue , and are corrected spherically for either two or three wavelengths see Table 1. Apochromatic objectives are the best choice for color photomicrography in white light. Because of their high level of correction, apochromat objectives usually have, for a given magnification, higher numerical apertures than do achromats or fluorites. Many of the newer high-performance fluorite and apochromat objectives are corrected for four dark blue, blue, green, and red or more colors chromatically and four colors spherically.

All three types of objectives suffer from pronounced field curvature, thus they project curved images rather than flat ones. Such artifact increases in severity with higher magnification. To overcome this inherent condition, optical designers have produced flat-field corrected objectives, which yield images that are in common focus throughout the viewfield.

Objectives that have flat-field correction and low distortion are called plan achromats, plan fluorites, or plan apochromats, depending upon their degree of residual aberration. This correction, although expensive, is extremely valuable in digital imaging and conventional photomicrography.

For many years, field curvature went uncorrected as the most severe optical aberration that occurred in fluorite semi-apochromat and apochromat objectives, tolerated as an unavoidable artifact. The introduction of flat-field plan correction to objectives perfected their use for photomicrography and video microscopy, and today these corrections are standard in both general use and high-performance objectives.

Figure 3 illustrates how correction for field curvature for a simple achromat adds a considerable number of lens elements to the objective. The significant increase in lens elements for plan correction also occurs with fluorite and apochromat objectives, frequently resulting in an extremely tight fit of lens elements see Figure 1 within the internal objective sleeve.

Older objectives typically have lower numerical apertures, and are subject to chromatic difference of magnification, an aberration that requires correction by the use of specially designed compensating oculars or eyepieces.

This type of correction was prevalent during the popularity of fixed tube length microscopes, but is not necessary with modern infinity-corrected objectives and microscopes. Recently, correction for chromatic difference of magnification is either built into the modern microscope objectives themselves Olympus and Nikon , or corrected in the tube lens Leica and Zeiss. The intermediate image in an infinity-corrected system appears behind the tube lens in the optical pathway at the reference focal length.

The tube lens focal length varies between and millimeters, depending upon design constraints imposed by the manufacturer. By dividing the reference focal length by the focal length of the objective lens, the magnification of an infinity-corrected objective can be calculated.

In many biological and petrographic applications, when mounting the specimen, a glass coverslip is used to both protect the integrity of the specimen and to provide a clear window for observation. The coverslip acts to converge the light cones originating from each point in the specimen.

But it also introduces chromatic and spherical aberration that must be corrected by the objective. The refractive index, dispersion, and thickness of the coverslip determine the degree to which light rays are converged. An additional concern is the aqueous solvent or excess mounting medium that lies between the specimen and coverslip in wet or thickly mounted preparations, which add to the variations in refractive index and thickness of the cover slip.

The imaging medium between the objective front lens and the specimen cover slip is another important element in respect to correction for spherical aberration and coma in the design of lens elements for objectives. Lower power objectives are designed to be used with only air as the imaging medium between the objective front lens and the coverslip.

The maximum theoretical numerical aperture obtainable with air is 1. The effect of coverslip thickness variation is negligible for dry objectives having numerical apertures less than 0. It is possible to correct for variations in coverslip thickness. For many applications a long free working distance is highly desirable and often necessary , and specialized objectives are designed for such use despite the difficulty involved in achieving large numerical apertures and the necessary degree of correction for optical aberrations.

Long working distance objectives are particularly useful when examining specimens in vitro through thick glass walls and for chemical and metallurgical microscopy, where the objective front lens must be protected against environmental hazards such as heat, caustic vapors, and volatile chemicals by a thick coverslip. The working distance of these objectives often exceeds two to three times that of comparable objectives having the same or a slightly greater numerical aperture.

Note that working distance decreases with magnification and numerical aperture, but not as dramatically as the objectives listed in Table 1. Also note that the SLWD objectives exhibit significantly longer working distances, but correspondingly lower numerical apertures, than the ELWD series of objectives.

It has become practical with modern manufacturing techniques to considerably improve the mechanical precision of microscope objectives, including their centration and parfocal distance, the distance between the specimen plane and the shoulder of the flange by which the objective lens is supported on the revolving nosepiece see Figure 1.

Thus, on modern research-grade microscopes, the specimen can be kept quite closely in focus within a micron or so , as well as centered in the field of view, when one turns the revolving nosepiece and switches from one objective to another.

For many years, objectives designed for biological applications from most manufacturers all conformed to an internationally recognized convention, a parfocal distance of