Setting The Transmitted Light Phase Contrast - Ryf Axio Lab.A1 Operating Manual

Carl Zeiss
Fig. 4-3 Center darkfield stop on condenser,
achromatic-aplanatic 0.9 H D Ph DIC
4.1.3

Setting the transmitted light phase contrast

(1) General principle
The phase contrast technique is ideal for examining thin, unstained specimens such as cultured cells.
Generally, the human eye is unable to perceive phase differences (index and thickness differences)
between the different cell components.
The phase contrast technique uses "phase stop and phase ring" optical modulators and interference
procedures in forming the intermediate image in order to transform small phase differences into
differences in intensity and color which are visible to the human eye.
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.
Interference in the intermediate image level occurs due to the differently influenced partial beams, and
these strengthen or weaken according to phase position. This interference results mainly in image
contents displaying differences in intensity which can be perceived by the human eye.
(2) Instrumentation
All Axio Lab.A1 microscopes, except stands for reflected light, are suitable for phase contrast
applications.
Phase contrast objectives with phase rings Ph 1, Ph 2 or Ph 3 for different average numerical
apertures which can also be used in the brightfield.
Condenser with turret/modulator disk containing centering phase stops Ph 1, Ph 2 and Ph 3 for
different average numerical apertures.
The phase stop used on the condenser must correspond to the label on the objective used,
e.g. Ph 1.
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OPERATION
Lighting and contrasting method in transmitted light
430037-7144-001
Reinsert the eyepiece into the tube.
If the height of the darkfield condenser is set
correctly and sensitively, it is possible to reduce
any brightening in the field of view left, and the
luminous-field diaphragm image appears almost
perfectly in focus.
Finally, match the size of the luminous-field
diaphragm to the size of the field of view.
Dark field microscopy requires specimens to be
considerably cleaner than in other techniques. In
particular fingerprints, dirt or dust particles have a
negative effect, as they brighten the background
of the field of view and decrease the contrast of
the object image.
Axio Lab.A1
04/2013