Page 26 - Fundamentals of Light Microscopy and Electronic Imaging

ADJUSTING THE MICROSCOPE FOR KOEHLER ILLUMINATION 9

• Preliminaries. Place a specimen slide, such as a stained histological specimen, on
the stage of the microscope. Adjust the condenser height with the condenser focus-
ing knob so that the front lens element of the condenser comes within 1–2 mm of
the specimen slide. Do the same for the objective lens. Be sure all diaphragms are
open so that there is enough light (including the illuminator’s field diaphragm, the
condenser’s front aperture diaphragm, and in some cases a diaphragm in the objec-
tive itself). Adjust the lamp power supply so that the illumination is bright but com-
fortable when viewing the specimen through the eyepieces.
• Check that the lamp fills the front aperture of the condenser. Inspect the front aper-
ture of the condenser by eye and ascertain that the illumination fills most of the
aperture. It helps to hold a lens tissue against the aperture to check the area of illu-
mination. Using an eyepiece telescope or Bertrand lens, examine the back aperture
of the objective and its conjugate planes, the front aperture of the condenser, and the
lamp filament. Be sure the lamp filament is centered, using the adjustment screws
on the lamp housing if necessary, and confirm that the lamp filament is focused in
the plane of the condenser diaphragm. This correction is made by adjusting the
focus dial of the collector lens on the lamp housing. Once these adjustments are
made, it is usually not necessary to repeat the inspection every time the microscope
is used. Instructions for centering the lamp filament or arc are given in Chapter 3.
Lamp alignment should be rechecked after the other steps have been completed.
• Focus the specimen. Bring a low-power objective to within 1 mm of the specimen,
and looking in the microscope, carefully focus the specimen using the microscope’s
coarse and fine focus dials. It is helpful to position the specimen with the stage con-
trols so that a region of high contrast is centered on the optic axis before attempting
to focus. It is also useful to use a low magnification dry objective (10–25 , used
without immersion oil) first, since the working distance—that is, the distance
between the coverslip and the objective—is 2–5 mm for a low-power lens. This
reduces the risk of plunging the objective into the specimen slide and causing dam-
age. Since the lenses on most microscopes are parfocal, higher magnification
objectives will already be in focus or close to focus when rotated into position.

Figure 1-5
August Koehler introduced a new method of illumination that greatly improved image quality
and revolutionized light microscope design. Koehler introduced the system in 1893 while he
was a university student and instructor at the Zoological Institute in Giessen, Germany,
where he performed photomicrography for taxonomic studies on limpets. Using the
traditional methods of critical illumination, the glowing mantle of a gas lamp was focused
directly on the specimen with the condenser, but the images were unevenly illuminated and
dim, making them unsuitable for photography using slow-speed emulsions. Koehler’s
solution was to reinvent the illumination scheme. He introduced a collector lens for the lamp
and used it to focus the image of the lamp on the front aperture of the condenser. A
luminous field stop (the field diaphragm) was then focused on the specimen with the
condenser focus control. The method provided bright, even illumination, and fixed the
positions of the focal planes of the microscope optics. In later years, phase contrast
microscopy, fluorescence microscopy with epi-illumination, differential interference contrast
microscopy, and confocal optical systems would all utilize and be critically dependent on the
action of the collector lens, the field diaphragm, and the presence of fixed conjugate focal
planes that are inherent to Koehler’s method of illumination. The interested reader should
refer to the special centenary publication on Koehler by the Royal Microscopical Society (see
Koehler, 1893).