OPERATION
Axioscope 5/7/Vario
Illumination and contrast methods in transmitted light …
ZEISS
03/2018
430035-7344-001
93
4.2.3
Setting up transmitted light phase contrast microscopy
(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 uses optical modulators such as the “annular phase diaphragm and phase
ring” to convert the small phase differences in intensity and color differences which are visible to the
human eye. The interference of different beams in the intermediate image is important for the
generation of such images.
With the aid of the optically defined ring channel "annular phase diaphragm and phase ring", the high-
intensity direct light portions are attenuated 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 are thus differently affected and achieve interference
and strengthen or weaken each other (constructive and destructive interference) – 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 PhC 1, PhC 2 or PhC 3 for various medium numeral
apertures which can also be used in the brightfield.
−
Condenser with revolver/modulator disk which contains centerable ring diaphragms PhC 1, PhC 2
and PhC 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. PhC 1.
(3) Setting up transmitted light phase contrast microscopy
•
Swivel 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.
