Microscope

The following is intended to acquaint the student with the light microscope. The major parts of the instrument will be named and a method for the effective use of the microscope will be outlined. The comments apply to the Nikon student microscope but will apply directly, or with slight modification, to student instruments of other manufacturers.

The microscope consists of a compound optical system (the objective lens and the ocular lens); a movable specimen support (the mechanical stage); an illumination system (the lamp and the condenser lens with its iris diaphragm). All the systems are attached to the microscope stand consisting of the base and arm.

The microscopes used in the course have a binocular head, which may be rotated after loosening its clamping screw. The topmost elements in the optical system are the eyepieces or ocular lenses. The interpupillary distance may be varied. One of the eyepieces may have a pointer, and note that one (or both) eyepiece(s) may be focused separately to compensate for dioptric differences between your eyes.

The revolving nosepiece is the inclined, circular metal plate to which the objective lenses, usually four, are attached. The objective lenses usually provide 4x, 10x, 40x and 100x magnification. The final magnification is the product of the magnification of the ocular and objective lenses. A slide is placed on the mechanical stage and is moved by rotating the stage control knobs. The lamp is an integral part of the base. The fine and course focus controls are mounted coaxially on the stand just above the base. The arm connects the base to the binocular head-revolving nosepiece assembly. The component of the illumination system immediately below the stage is the condenser. The height of the condenser may be varied to give a bright, evenly illuminated field. Generally the condenser is used in its highest position or just slightly lower. A lever projects from the condenser and it is used to vary the opening of the condenser (or iris) diaphragm. For work with the scanning (4x) and low-power (10x) objectives, the condenser diaphragm should be wide open. For work with the high-dry (40x) and oil-immersion objectives (100x), however, the diaphragm should be closed slowly while looking at a sharply focused section until the level of illumination is just slightly reduced. This is the setting of the condenser diaphragm for optimum contrast and resolution. (From a theoretical point of view this is not quite correct. The diaphragm should be adjusted for each magnification. In most instances, however, it is much less critical at the lower magnifications).

In examining a slide with the light microscope, the following sequence of steps should be used:

1. Place the slide on the stage and examine it with the scanning objective (4x). Scan the entire section. Often tissue and organ identification can be made at this magnification. Select an area or areas for study at higher magnification.
2. Rotate the revolving nosepiece to place the lower-power objective (10x) in the optical axis. When turning the nosepiece, grasp the nosepiece itself or the part of the objective adjacent to the nosepiece to avoid excess stress on the objective.
3. Proceed to the next step in magnification, if necessary, which is high dry (40x). Adjust the condenser.
4. For some specimens, especially blood and cellular organelles, you may want to use the highest magnification, which is oil immersion (100x). The following procedure must be used when working with the oil immersion lens:
   1. focus carefully on a selected area with the high-dry objective,
   2. swing the high-dry objective out of the light path and allow the nosepiece to remain in an intermediate position between the high-dry and the oil-immersion objectives,
   3. place a drop of immersion oil (available in the bookstore) on the slide in the appropriate region to be studied,
   4. swing the oil-immersion objective into position. The distance between the front element of the objective and the surface of the slide will be about 1.0mm, and the oil will form a bridge between the slide and the objective. The area to be examined should be within the field and should require only slight refocusing. You may need to readjust the condenser.

When finished using the oil-immersion objective, both the objective and the slide must be wiped with lens paper (available in the bookstore). If oil is allowed to dry on the high-dry or oil-immersion objective, the optical performance of the instrument will be severely reduced. The dried oil must be removed with lens paper.

Be careful not to move the high-dry objective into position while oil is on the slide. If this is done by mistake, the high-dry objective must be cleaned by wiping the front element with lens paper.

Slides should be cleaned with lens paper or tissue to remove fingerprints, oil or dirt. If the slide cannot be cleaned with a dry tissue, use alcohol.