OPERATION
Carl Zeiss
Illumination and contrast methods
Axio Imager
148
430000-7344-001
M70-2-0020 e 06/2009
4.9.2
Setting transmitted-light darkfield
(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 changes markedly if such specimens
are viewed in transmitted-light dark field, where the specimen is illuminated using an aperture that 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) Instrument
configuration
−
Universal condenser with dark-field 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 by analogy with transmitted-light brightfield. However, use the objective with
the highest aperture instead of the 10x objective.
•
Turn the turret disk 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 (4-116/
1
and
4
) to turn the two
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 when driving against a mechanical
resistance. After having removed 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 has been perfectly set, when the background of the field of view appears as dark
as possible.