OPERATIONZEISS Illumination and contrast methods in reflected light … Axioscope 5/7/Vario122 430035-7344-001 03/20184.3.6 Setting up reflected light fluorescence(1) General principle of operationThe reflected light fluorescence method is used to show fluorescent substances in typical fluorescentcolors in high contrast. The light originating from a high-performance illuminator in a reflected lightfluorescence microscope passes through a heat protection filter onto an excitation filter (bandpass). Thefiltered short-wave excitation radiation is reflected by a dichroitic beam splitter and is focused on thesample through the objective. The sample absorbs the short-wave radiation before emitting longer-wavefluorescence radiation (Stokes’ Law). This radiation is then captured from the image side by the objectiveand 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 beinserted 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 thefilter slider is optional)− HBO 100 or Colibri 3 or HXP 120 illuminator for reflected light illumination− Fluorescence protection shieldNOTEThe mercury vapor short arc lamp must be adjusted before starting the reflected lightfluorescence method. Re-adjusting the adjustment might become necessary depending on thelength of operating time.(3) Setting up reflected light fluorescenceSetting the reflected light fluorescence is facilitated when starting with an objective of average degree ofmagnification, e.g. EC Plan-Neofluar 20x/0.50, and a sample of high fluorescence. The use ofdemonstration samples is also advisable for the start-up.NOTEIf necessary, remove the λ compensator used for the transmitted light polarization methodfrom 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 phasecontrast if using a PhC objective) and go to the sample detail to be examined.
