Zeiss Axio Scope.A1, Operating Manual

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OPERATION
Carl Zeiss Illumination and Contrasting Method Axio Scope.A1
74
M60-2-0007 e 05/08
4.1.3 Adjusting the Transmitted Light/Phase-Contrast
(1) General principle of operation
The phase-contrast method is ideal for examining thin uncolored samples, e.g. culture cells. Generally the
human eye cannot detect phase differences (variations in refractive index or thickness) within the
different cell components.
The phase-contrast method converts with the aid of optical modulators, such as "annular phase
diaphragm and phase ring", the small phase differences into intensity and color differences which are
visible to the human eye. Important for the generation of such images is the interference of different
beams in the intermediate image.
With the aid of the optically defined ring channel "annular phase diaphragm and phase ring", the high-
intensity direct light portions are damped and provided with a constant phase shift. The indirect light
portions, however, which are diffracted by different cell particles, bypass this optical channel and their
phase is affected by the difference in the sample’s refractive index and thickness.
In the intermediate image plane, the partial beams thus differently affected achieve interference and
strengthen or weaken each other – depending on their phase. As a result, these interferences create
image contents with intensity differences visible to the human eye.
(2) Instrumentation
−
Phase-contrast objectives with the phase rings Ph 1, Ph 2 or Ph 3 for various medium numeral
apertures which can also be used in the bright-field.
−
Condenser with revolver / modulator disk which contains centerable ring diaphragms Ph 1, Ph 2
and Ph 3 for various medium numeral apertures.
−
The phase annular diaphragm used on the condenser must be consistent with the labeling on the
objective which is used, e.g. Ph 1.
(3) Adjusting the transmitted light/phase-contrast
•
Put the phase-contrast objective, e.g. labeled Ph 1 , into the beam path.
•
Switch on the annular phase diaphragm on the condenser’s revolver disk which has the same label as
the phase-contrast objective, e.g. 1.
•
In order to check the centering and the overlap of the lighter annular diaphragm (in the condenser)
with the darker phase ring (in the objective), remove an eyepiece from the tube and replace it with an
auxiliary microscope. Use the adjusting fixture on the auxiliary microscope to focus the annular
diaphragm and the phase ring in the objective exit pupil.