OPERATION
Axio Examiner Illumination and contrast procedures Carl Zeiss
M60-2-0003 e 05/2012 67
4.1.5 Reflected-light fluorescence
(1) General operating principle
The reflected-light fluorescence method allows the high-contrast representation of fluorescent substances
in typical fluorescence colors. In a reflected-light fluorescence microscope, the light generated by a
powerful illuminator passes through a heat protection filter onto the excitation filter (band-pass filter).
Filtered shortwave exciting radiation is reflected by a dichromatic beam splitter, and focused on the
specimen by the objective. The specimen absorbs the shortwave radiation and then emits fluorescence
radiation with a longer wavelength (Stoke's law) which is captured by the objective on the image side,
and transmitted by the dichromatic beam splitter. Finally, the beams pass a barrier filter (long-pass/band-
pass filter) which only allows the passage of the long-wave radiation emitted by the specimen.
Excitation filter and barrier filter require a very precise spectral coordination between both of them, and
are located together with the corresponding dichromatic beam splitter in a FL P&C reflector module.
(2) Equipment
Every microscope allows the performance of the reflected-light fluorescence procedure.
− Recommended objectives: Plan-Neofluar or Fluar (UV excitation)
− FL P&C reflector module and shutter plate in the reflector turret
− Filter drawer A
− FL attenuator (only for Axio Examiner.D1/.Z1)
− HXP 120, HBO 100 or HBO 50 illuminator for reflected-light illumination
− HAL 100 halogen lamp for transmitted-light illumination
The mercury-vapor short-arc lamp must have been adjusted in every case before applying the
reflected-light fluorescence procedure (see sections 3.1.14.3 and 3.1.15.3).
A readjustment should be performed if necessary, depending on the operating time.
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