OPERATION
ZEISS
Illumination and contrast methods in reflected light
Axiolab 5
116
430037-7444-001
05/2019
4.3.4
Setting reflected light fluorescence
(1) General principle
The reflected light fluorescence method is used to show fluorescent substances in typical fluorescence
colors in high contrast. The light originating from a high-performance illuminator in a reflected light
fluorescence microscope passes through a heat protection filter to an excitation filter (bandpass). The
filtered, short-wavelength excitation beam is reflected by a dichroic beam splitter and is focused on the
specimen above the objective. The specimen absorbs the short-wave radiation before emitting longer-
wave fluorescence radiation (Stokes’ Law). This radiation is then captured from the image side by the
objective and passes through the dichroic beam separator. Finally, the beams pass through a band
elimination filter (longpass/bandpass) which only permits the long-wave radiation emitted by the
specimen to pass through.
The spectra of the excitation and the band-elimination filters must match very closely. They must be
inserted in a reflector module FL P&C together with the respective dichroic beam splitter.
Only powerful LEDs are supplied as FL excitation light sources in the Axiolab 5 program with the
following options:
LED module 385nm for Axio 423052-9593-000
LED module 470nm for Axio 423052-9573-000
LED module 505nm for Axio 423052-9562-000
LED module 565nm for Axio 423052-9602-000
LED module 625nm for Axio 423052-9522-000.
(2) Instrumentation
Observations in reflected light fluorescence can be made only on Axiolab 5 microscopes for reflected light
and reflected light fluorescence.
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Recommended objectives: EC Plan-Neofluar or Fluar (UV excitation)
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LED modules for FL excitation (installed in FL stand)
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FL P&C reflector modules equipped with respective filter sets
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Fluorescence protection shield
