Page 24 - Fundamentals of Light Microscopy and Electronic Imaging

ADJUSTING THE MICROSCOPE FOR KOEHLER ILLUMINATION 7

condenser. The size of the illuminated field at the specimen is adjusted so that it matches
the specimen field diameter of the objective lens being employed. Because each source
point contributes equally to illumination in the specimen plane, variations in intensity in
the image are attributed to the object and not to irregular illumination from the light
source. The method of illumination introduced by August Koehler fulfills these require-
ments and is the standard method used in light microscopy (Fig. 1-5). Under the condi-
tions set forth by Koehler, a collector lens on the lamp housing is adjusted so that it
focuses an image of the lamp filament at the front focal plane of the condenser while
completely filling the aperture; illumination of the specimen plane is bright and even.
Achieving this condition also requires focusing the condenser using the condenser focus
dial, an adjustment that brings two sets of conjugate focal planes into precise physical
locations in the microscope, which is a requirement for a wide range of image contrast-
ing techniques that are discussed in Chapters 7 through 12. The main advantages of
Koehler illumination in image formation are:

• Bright and even illumination in the specimen plane and in the conjugate image
plane. Even when illumination is provided by an irregular light source such as a
lamp filament, illumination of the object is remarkably uniform across an extended
area. Under these conditions of illumination, a given point in the specimen is illu-
minated by every point in the light source, and, conversely, a given point in the light
source illuminates every point in the specimen.
• Positioning of two different sets of conjugate focal planes at specific locations
along the optic axis of the microscope. This is a strict requirement for maximal spa-
tial resolution and optimal image formation for a variety of optical modes. As we
will see, focusing the stage and condenser positions the focal planes correctly,
while adjusting the field and condenser diaphragms controls resolution and con-
trast. Once properly adjusted, it is easier to locate and correct faults such as dirt and
bubbles that can degrade optical performance.

ADJUSTING THE MICROSCOPE FOR KOEHLER ILLUMINATION

Take a minute to review Figure 1-4 to familiarize yourself with the locations of the two
sets of focal planes: one set of four field planes and one set of four aperture planes. You
will need an eyepiece telescope or Bertrand lens to examine the aperture planes and to
make certain adjustments. In the absence of a telescope lens, you may simply remove an
eyepiece and look straight down the optic axis at the objective aperture; however, with-
out a telescope the aperture looks small and is difficult to see. The adjustment procedure
is given in detail as follows. You will need to check your alignment every time you
change a lens to examine a specimen at a different magnification.

Note: Summary of Steps for Koehler Illumination

1. Check that the lamp is focused on the front aperture of the condenser.
2. Focus the specimen.
3. Focus the condenser to see the field stop diaphragm.

4. Adjust the condenser diaphragm using the eyepiece telescope.