OPERATION
ZEISS
Illumination and contrast methods in transmitted light
Axiolab 5
84
430037-7444-001
05/2019
4.2.2
Configuring transmitted light darkfield microscopy using the KÖHLER method
(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 brightfield microscopy. This is radically changed when
such specimens are observed in transmitted light darkfield microscopy. In principle, the specimen is
exposed to light from an illumination aperture which is larger than that of the objective used.
In darkfield microscopy, only the diffracted and scattered light components which are important for
imaging reach the objective, while the direct unchanged light bundles are routed past the objective. This
is one of the reasons why even fine structures that are sometimes below the resolving power of the light
microscope can be resolved and appear very bright on a dark background.
(2) Instrumentation
All Axiolab 5 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 BF, DF, Ph 1, Ph 2, Ph 3
−
Condenser, achrom.-aplan. 0.9 H D Ph DIC
−
Darkfield condenser with dry darkfield (465505-0000-000 applicable aperture from 0.6 – 0.75)
−
Ultra condenser (465500-0000-000 applicable aperture from 0.75 – 1)
(3) Configuring transmitted light darkfield
microscopy
•
Adjust the brightness using the KÖHLER
method as for transmitted light brightfield
microscopy. 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.
•
Position the turret/modulator disk of the
condenser at
D
and swivel in the condenser
front lens (if existing).
•
Remove the eyepiece from the tube (or replace
it with an 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.
•
To center the darkfield stop (not possible with all condensers), use two Allen wrenches (AF 1.5)
(Fig. 4-5/
1
) to turn the two centering screws (Fig. 4-5/
2
and
3
) until the exit pupil of the objective
appears homogeneously dark. After centering, remove both Allen wrenches (AF 1.5) from the
condenser.
Fig. 4-5
Centering the darkfield stop on
condenser, achromatic-aplanatic 0.9
H D Ph DIC
