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OPERATION
Axio Imager
Illumination and contrast methods
Carl Zeiss
M70-2-0020 e 06/2009
430000-7344-001
151
4.9.3
Setting transmitted-light phase contrast
(1) General
principle
The phase contrast technique is ideal for examining thin, unstained specimens, e.g. culture cells. The
human eye is unable to see phase differences (differences in refractive index and thickness) between the
different cell components.
The phase contrast technique uses the optical modulators "phase stop and phase ring" as well as the
interference procedures during formation of the intermediate image in order to change the small phase
differences into intensity and color differences that are visible to the eye.
The high-intensity, direct light components are attenuated with the optically defined ring channel "phase
stop and phase ring" and given a constant phase shift. The indirect light components diffracted at
different cell components, however, bypass this optical channel and are influenced in phase by the
refractive index and the thickness differences in the specimen.
In the intermediate image plane, the thus differently influenced partial beams interfere with each other
and are amplified or attenuated - depending on the phase position. This interference results in image
contents displaying differences in intensity and color that can be perceived by the human eye.
(2) Instrument
configuration
−
Phase-contrast objectives with phase rings Ph 1, Ph 2 or Ph 3 for different average numerical apertures
that can also be used in brightfield without any restriction.
−
Universal condenser with turret disk containing centerable phase stops Ph 1, Ph 2 and Ph 3 for
different average numerical apertures.
−
The phase stop on the universal condenser swiveled into the light path must agree with the
corresponding label on the objective, e.g. Ph 1.
(3)
Setting transmitted-light phase contrast
•
Swivel phase-contrast objective, e.g. the one labeled with Ph 1, into the light path.
•
On the turret disk of the universal condenser, swivel in the phase stop labeled like the phase-contrast
objective, e.g. Ph 1.
•
To check centering and congruence of the bright phase stop (in the condenser) with the dark phase
ring (in the objective), remove one eyepiece from the tube and replace it with the auxiliary microscope.
Use the correction device on the auxiliary microscope to focus on the phase stop and the phase ring in
the exit pupil of the objective.
To check the centration, you can also use the Bertrand lens slider PH. However, this is possible
only if there is no camera path deflection installed on the left side of the microscope stand.