(3) Regulation of the amount of light admitted is accomplished by the iris
diaphragm in the substage condenser. The size of the opening in the diaphragm is
controlled by a lever on the side of the condenser. The lever of the iris diaphragm
should never be forced to the full limit in either direction. Doing so may damage the
delicate leaves of the diaphragm. Generally, when observing liquid preparations under
low power, the diaphragm opening should be partially closed. Under the high dry
objective, the diaphragm is generally opened to a greater degree to allow more light to
pass through the material. When observing stained preparations under the oil
immersion objective, the iris diaphragm is usually opened wide.
(4) The substage condenser functions to direct a light beam of the desired
numerical aperture (N.A.) and field size onto the specimen. The size of the opening in
the condenser together with its position up or down controls the light entering the
system. When the condenser is close to the stage, concentration of light is greater; as
the condenser is moved downward, less light passes upward through the object under
(5) Improper illumination is indicated when: (1) dark points or shadows
appear in the field; (2) the outline of an object is bright on one side and dark on the
other; or (3) the object appears to be in a glare of light. This can usually be corrected
by changing the position of the mirror, by reducing the amount of light by adjusting the
size of the opening in the iris diaphragm, or by raising or lowering the condenser.
d. Focusing. Focusing can be defined as the adjustment of the relationship
between the optical system and the object so that a clear image is obtained. Several
important rules to be observed when focusing the microscope on the preparation are:
(1) After the object is mounted on the stage, the objective to be used is
turned into line with the eyepiece.
(2) Movement of the objective is accomplished by revolving the nosepiece.
The nosepiece is provided in order to enable rapid, convenient substitution of one
objective for another. This change is effected by grasping two of the objectives
between the thumb and forefinger of the right hand and rotating them until the desired
objective is brought into line with the axis of the body tube. It is very important that
exact alignment be obtained. The correct setting is indicated by a slight "click" as the
objective comes into position.
(3) Whenever the nosepiece is revolved, its movements should be observed
to make certain that the objectives do not come into contact with the object. Some
microscopes are not parfocal; that is, objects in focus under low power will not be in
focus when the nosepiece is rotated to a higher power of magnification. It may,
therefore, be necessary to refocus when changing to higher magnification. In
microscopes that are parfocal, it is possible to swing other objectives into place without
touching the coarse adjustment and with only a slight turn of the fine adjustment knob
required to restore perfect focusing.