OPERATION
ZEISS
Illumination and contrast methods in reflected light …
Axioscope 5/7/Vario
122
430035-7344-001
03/2018
4.3.6
Setting up reflected light fluorescence
(1) General principle of operation
The reflected light fluorescence method is used to show fluorescent substances in typical fluorescent
colors in high contrast. The light originating from a high-performance illuminator in a reflected light
fluorescence microscope passes through a heat protection filter onto an excitation filter (bandpass). The
filtered short-wave excitation radiation is reflected by a dichroitic beam splitter and is focused on the
sample through the objective. The sample 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 dichroitic beam separator. Last, the beams pass through a band-elimination filter
(longpass/bandpass) and only the long-wave radiation emitted by the sample passes.
The spectra of the excitation and the band-elimination filters must match very closely. They must be
inserted in a FL P&C reflector module together with the according dichroitic beam splitter.
(2) Instrumentation
−
Appropriate objectives, e.g. EC Plan-Neofluar or Fluar (UV-excitation)
−
FL P&C reflector module and safety shutter in the upper part (additional shutter function with the
filter slider is optional)
−
HBO 100 or Colibri 3 or HXP 120 illuminator for reflected light illumination
−
Fluorescence protection shield
NOTE
The mercury vapor short arc lamp must be adjusted before starting the reflected light
fluorescence method. Re-adjusting the adjustment might become necessary depending on the
length of operating time.
(3) Setting up reflected light fluorescence
Setting the reflected light fluorescence is facilitated when starting with an objective of average degree of
magnification, e.g. EC Plan-Neofluar 20x/0.50, and a sample of high fluorescence. The use of
demonstration samples is also advisable for the start-up.
NOTE
If necessary, remove the
λ
compensator used for the transmitted light polarization method
from its compartment before setting the reflected light fluorescence.
•
Slide the fluorescence protection shield in the compensator slot above the nosepiece.
•
Switch on the objective EC Plan-Neofluar 20x/0.50.
•
Turn the condenser turret to the BF transmitted light brightfield position first (or choose phase
contrast if using a PhC objective) and go to the sample detail to be examined.
