Operation
Carl Zeiss Setting epi-fluorescence Axiostar plus
3-14 B 40-81 e 12/01
3.6 Setting epi-fluorescence
3.6.1 General principle
The epi-fluorescence method displays
fluorescent substances with strong contrast in
typical fluorescent colors. In an epi-fluorescence
microscope, this occurs through a light
generated via a heat protection filter on the
excitation filter (bandpass). The filtered short-
wave excitation beam is reflected by a
dichromatic beam splitter and focused on the
specimen via the objective. The specimen
absorbs the short-wave beam and emits a long-
wave fluorescence beam (Stoke’s Law), which is
registered by the objective and let through by
the dichromatic beam splitter. Then the beams
pass through a blocking filter
(lowpass/bandpass) that only allows long-wave
beams emitted from the specimen to pass.
The excitation and blocking filters must be
calibrated spectrally; they are both in the
reflector module FL P&C, along with the
dichromatic beam splitter.
You can get an overview of filter sets and
fluorochromes available from Zeiss at:
www.zeiss.de/micro
“Fluorescent microscopy” under
“Techniques of Microscopy”
3.6.2 Epi-fluorescence configuration
• Recommended objectives Plan-Neofluar or
Fluar (UV excitation).
• Epi-fluorescence illumination with reflector
module FL P&C.
• Mercury vapor short-arc lamp HBO 50 for
incident illumination.
☞
The mercury vapor short-arc lamp must be
adjusted with the adjustment guide before
using it for the epi-fluorescence procedure. If
necessary, you must readjust the setting
depending on the amount of use.
3.6.3 Setting epi-fluorescence
Before starting:
– The microscope is ready for use as outlined
in section 2.
Settings:
• Turn on the halogen lamp with the line
switch on the microscope.
• Swivel the desired objective into place.
• Search for the specimen location to be
observed in the transmitted light. If the 5-
position Abbe condenser with turret is to be
used, set the turret to position H
transmitted-light brightfield (or phase
contrast).
• Set KÖHLER illumination in the same way as
transmitted-light brightfield.
• Focus on the specimen with the focusing
drive.
• Turn off halogen lamp.
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