Zeiss Axioscope 7, Руководство по эксплуатации, Страница: 90 / 140

OPERATION
ZEISS Illumination and contrast methods in transmitted light … Axioscope 5/7/Vario
90 430035-7344-001 03/2018
4.2.2 Setting up transmitted light darkfield microscopy using the KÖHLER method
(1) General principle of operation
Uncolored biological samples, like bacteria or living cell cultures, are sometimes hard to see in transmitted
light brightfield microscopy due to their translucence. Yet it is completely different when you examine
these samples with the transmitted light darkfield method. You basically illuminate the sample with an
illumination aperture which is higher than the one of the objective you are using.
In darkfield microscopy, 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 BF, DF, PhC 1, PhC 2, PhC 3,
−
Condenser, achrom.-aplan. 0.9 BF DF PhC 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) Setting up transmitted light darkfield
microscopy
•
Adjust the KÖHLER illumination as for
transmitted light brightfield microscopy. Instead
of the 10x objective it is necessary to use an
objective with the highest possible aperture.
The aperture must not exceed the aperture of
the condenser being used.
•
Set the revolver/modulator disks to position
D
and bring the condenser front lens into position
(if applicable).
•
Take the eyepiece out of the tube (or replace it
with the auxiliary microscope) and check the
centering of the darkfield diaphragm in the
objective exit pupil. If the DF central darkfield
diaphragm in the universal condenser is partially
outside or is not centered to the objective exit
pupil and the pupil does not appear consistently
dark, it is necessary to re-adjust the centering of
the darkfield diaphragm.
•
If the darkfield diaphragm needs to be centered, use two Allen wrenches (AF 1.5) (
630H
Fig. 4-6/
1
) and
adjust the two centering screws (
631H
Fig. 4-6/ 2 and 3 ) until the objective exit pupil appears consistently
dark. Remove the Allen wrenches (AF 1.5) from the condenser after finishing the centering procedure.
Fig. 4-6 Centering the darkfield diaphragm
on the condenser, achromatic-
aplanatic 0.9 H D PhC DIC