OPERATION
Axiolab 5
Illumination and contrast methods in transmitted light
ZEISS
05/2019
430037-7444-001
87
4.2.3
Configuring transmitted light phase contrast microscopy
(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. However, the indirect light components diffracted
at different cell components 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 Axiolab 5 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.
(3) Configuring 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.
