OPERATION
Axio Lab.A1
Lighting and contrasting method in transmitted light
Carl Zeiss
04/2013 430037-7144-001
75
4.1.2
Adjusting the transmitted light darkfield according to KÖHLER
(1) General principle
Due to their transparency, unstained biological specimens, such as bacteria or living cell cultures, are
often barely or not at all visible in transmitted light bright field. This is radically changed when such
specimens are observed in the transmitted light darkfield. In principle, the specimen is exposed to light
from an illumination aperture which is larger than that of the objective used.
In dark field, only the diffracted and scattered light components, which are important for imaging, reach
the objective, while the direct unchanged light bundles are directed past the objective. This is one of the
reasons why even fine structures can be resolved, which are sometimes below the resolving power of the
light microscope and which appear very bright on a dark background.
(2) Instrumentation
All Axio Lab.A1 microscopes, except stands for reflected light, are suitable for darkfield applications.
Condenser with darkfield stop in position
D
e.g.:
Condenser 0.9/1,25 H with modulator disk H, D, Ph 1, Ph 2, Ph 3,
Condenser, achrom.-aplan. 0.9 H D Ph DIC,
Darkfield condenser with dry darkfield,
Ultra condenser.
(3) Adjusting the transmitted light darkfield
x
Adjust the brightness according to KÖHLER analogously to the method for the transmitted light
brightfield. Instead of the 10x objective, however, swivel in the objective with the highest aperture
which does not exceed the limit aperture for the darkfield with the condenser used.
x
Position the turret/modulator disk of the condenser at
D
and swivel in the condenser front lens (if
existing).
x
Remove the eyepiece from the tube (or replace with auxiliary microscope) and check the centering of
the darkfield diaphragm in the exit pupil of the objective. If the central darkfield stop D in the universal
condenser is partly outside of or de-centered to the exit pupil of the objective, and if the exit pupil is
not homogeneously dark, the darkfield stop must be re-centered.
x
To center the darkfield stop (not possible with all condensers), use the two SW 1.5 Allen screwdrivers
(Fig. 4-3/
1
and
4
) to turn the two centering screws (Fig. 4-3/
2
and
3
) until the exit pupil of the objective
appears homogeneously dark. After centering, remove both SW 1.5 screwdrivers from the condenser.
Since the apertures of objectives with an integrated aperture iris stop are too high for
transmitted light dark field, the aperture iris stop must at least be closed to the limit aperture
of 0.65.
The performance criterion for the darkfield method is always the darkest possible background of the field
of view.