ZEISS
OPERATION
Axio Imager 2
Illumination and contrast methods
164
430000-7544-001
01/2016
4.12.2
Setting the transmitted-light darkfield
(1)
General principle
Due to their transparency, unstained biological specimens, such as bacteria or living cell cultures, are
often barely visible (or not at all) in the transmitted-light brightfield. This changes significantly if such
specimens are observed in the transmitted light darkfield. In principle the specimen is illuminated with an
illumination aperture which is greater than that of the objective used.
In the darkfield, 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
reason 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)
Instrument equipment
−
Universal condenser with darkfield stop in position D
−
Achromatic-aplanatic universal condenser 0.9 H/0.8-0.9 DF (424216-0000-000), for low
magnifications with wide-field-DF-slider for 2.5x-5x (424215-0000-000)
−
Use of ICS objectives up to the maximum aperture of 0.75. Objectives with a higher aperture can be
used in conjunction with the above universal condenser only in the equipment version with an
integrated aperture iris stop and/or the screw-on darkfield attachment 1.2–1.4 Oil (424218-0000-
000).
(3)
Setting transmitted-light darkfield
•
Set KÖHLER illumination in the same way as for transmitted-light brightfield. However, use the
objective with the highest aperture instead of the 10x objective.
•
Turn the MKM of the universal condenser to position D and swivel in the condenser front lens.
•
Remove the eyepiece from the tube (or replace it with the auxiliary microscope) and check the
centering of the darkfield stop in the exit pupil of the objective. If the central darkfield stop D in the
universal condenser is outside of or decentered to the exit pupil of the objective, and if the exit pupil is
not homogeneously dark, the darkfield stop must be recentered.
•
To center the darkfield stop, use the two SW 1.5 Allen screwdrivers (Fig. 193/
1
and
4
) to turn the two
centering screws (Fig. 193/
2
and
3
) until the exit pupil of the objective appears homogeneously dark.
After centering, remove both SW 1.5 screwdrivers from the condenser.
Leaving the two Allen screwdrivers unintentionally in the motorized universal condenser does not pose a
risk to the drives of the condenser as these instantly switch off if a mechanical resistance is encountered.
After removing the source of the resistance, you can continue using the condenser as usual.
Since the apertures of objectives with an integrated aperture iris stop are too high for
transmitted-light dark field, the aperture iris stop must be closed to the limit aperture of 0.65.
The darkfield method is correctly set when the background of the field of view appears as dark as
possible.
