OPERATION
Axiolab 5
Illumination and contrast methods in transmitted light
ZEISS
05/2019
430037-7444-001
81
4.2
Illumination and contrast methods in transmitted light
4.2.1
Configuring transmitted light brightfield microscopy using the KÖHLER method
(1) General principle of operation
Transmitted light brightfield microscopy is the most commonly used method of optical microscopy. High-
contrast or tinted (stained) samples (e.g. a blood smear) can be examined easily and quickly.
For an imaging result which is as true to the specimen as possible, we must consider the so-called direct
bundled beams as well as the indirect ones, i.e. the beams which diffract and scatter on the sample
details. According to ABBE, the image is truer to the specimen when the fraction of the cone of light is as
large as possible.
The best performance of the microscope, and especially its objective, is achieved when the condenser,
field diaphragm and aperture diaphragm are adjusted using the KÖHLER illumination method. These
basic rules for adjusting a microscope are explained in detail in section 4.2.1 (3) "Configuring transmitted
light brightfield microscopy using the KÖHLER method".
(2) Instrumentation for transmitted light brightfield microscopy
The equipment of every Axiolab 5 microscope, except the stand for reflected light, permits the use of
transmitted light brightfield microscopy.
All available condensers, except special condensers such as darkfield condensers, can be used for
transmitted light brightfield microscopy.
(3) Configuring transmitted light brightfield microscopy using the KÖHLER method
−
The Axiolab 5 has been started up correctly (see section 3).
−
The Axiolab 5 is switched on.
•
Adjust the image brightness using the Intensity/LM knob (Fig. 4-3/
1
) on the microscope stand.
•
Insert a high-contrast specimen into the specimen holder of the mechanical stage.
•
If condensers with a swiveling front lens are used, swivel these into the beam path with
≥
10x
objectives and set the condenser with the knurled knob for vertical adjustment (Fig. 4-3/
3
or
2
) to the upper stop. The stop must be adjusted so that the specimen is not lifted by the
condenser (to set the condenser stop, see section 4.2.1 (4)).
•
When using condensers with a turret/modulator disk and knurled ring (Fig. 4-4/
3
), set position
B
(or
H
= brightfield).
•
Swivel in the 10x objective on the nosepiece (Fig. 4-3/
6
) and use the focus drive to focus on the
specimen (Fig. 4-3/
2
).
