OPERATIONZEISS Illumination and contrast methods in reflected light Axiolab 5116 430037-7444-001 05/20194.3.4 Setting reflected light fluorescence(1) General principleThe reflected light fluorescence method is used to show fluorescent substances in typical fluorescencecolors in high contrast. The light originating from a high-performance illuminator in a reflected lightfluorescence microscope passes through a heat protection filter to an excitation filter (bandpass). Thefiltered, short-wavelength excitation beam is reflected by a dichroic beam splitter and is focused on thespecimen 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 theobjective and passes through the dichroic beam separator. Finally, the beams pass through a bandelimination filter (longpass/bandpass) which only permits the long-wave radiation emitted by thespecimen to pass through.The spectra of the excitation and the band-elimination filters must match very closely. They must beinserted 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 thefollowing options:LED module 385nm for Axio 423052-9593-000LED module 470nm for Axio 423052-9573-000LED module 505nm for Axio 423052-9562-000LED module 565nm for Axio 423052-9602-000LED module 625nm for Axio 423052-9522-000.(2) InstrumentationObservations in reflected light fluorescence can be made only on Axiolab 5 microscopes for reflected lightand reflected light fluorescence.− Recommended objectives: EC Plan-Neofluar or Fluar (UV excitation)− LED modules for FL excitation (installed in FL stand)− FL P&C reflector modules equipped with respective filter sets− Fluorescence protection shield
