Zeiss Axio Scope.A1, Operating Manual

Page: 72 / 123

OPERATION
Carl Zeiss Illumination and Contrasting Method Axio Scope.A1
72
M60-2-0007 e 05/08
4.1.2 Adjusting the Transmitted Light/Dark-Field According to KÖHLER
(1) General principle of operation
Uncolored biological samples, like bacteria or living cell cultures, are sometimes hard to see in the
transmitted light/bright-field due to their translucence. Yet it is completely different when you examine
these samples with the transmitted light/dark-field method. You basically illuminate the sample with an
illumination aperture which is higher than the one of the objective you are using.
In the dark-field, only the diffracted and scattered light portions which are important for the imaging
procedure get into the objective, whereas the indirect unaffected light beams are directed past the
objective. Thus a resolution of fine structures is achieved which is partially below the resolution capacity
of a light microscope. The fine structures now appear bright and incandescent on a dark background.
(2) Instrumentation
Condenser with transmitted light 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,
−
Dark-field condenser with dry dark-field,
−
Ultra condenser.
(3) Adjusting the transmitted light/dark-field
•
Adjust the illumination according to KÖHLER in analogy to the transmitted light/bright-field. Instead of
the 10x objective it is necessary to use a objective with the highest possible aperture. The aperture
must not exceed the border aperture of the dark-field with the used condenser.
•
Adjust revolver- / modulator disks to position D and bring in condenser front optic (if applicable).
•
Take eyepiece out of the tube (or replace it by the auxiliary microscope) and check the centering of the
dark-field diaphragm in the objective exit pupil. If the central dark-field diaphragm D in the universal
condenser lies partially outside or is not centered to the objective exit pupil and the pupil does not
appear consistently dark, then it is necessary to re-adjust the centering of the dark-field diaphragm.
•
If the dark-field diaphragm needs to be centered, please use both Allen screwdrivers SW 1.5
(
630H630H
Fig. 4-4/ 1 and 4 ) and adjust the two centering screws (
631H631H
Fig. 4-4/ 2 and 3 ) until the objective exit pupil
appears consistently dark. Remove the Allen screwdrivers SW 1.5 from the condenser after finishing
the centering procedure.
Objectives with built-in iris apertures have apertures which are too high for the transmitted
light/dark-field. Stop the iris aperture down at least until it reaches the border aperture for the
dark-field of the condenser in use.
Performance criterion for the dark-field method is always a back ground for the field of view which
should be as dark as possible