Zeiss Axio Scope.A1 Operating Manual

Contents

OPERATIONAxio Scope.A1 Illumination and Contrasting Method Carl ZeissM60-2-0007 e 05/08 934.1.12 Adjusting Reflected Light Fluorescence(1) General principle of operationThe 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 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 reflector module FL P&C together with the according dichroitic beam splitter.(2) Instrumentation− Appropriate objectives, e.g. EC Plan-Neofluar or Fluar (UV-excitation)− Reflector module FL P&C and safety shutter in the upper part (additional shutter function with thefilter slider is optional)− Lamp HBO 100 or HBO 50, Colibri or HXP 120 for reflected light illumination for reflectedillumination− Fluorescence protection shieldThe 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) Adjusting reflected light fluorescenceAdjusting the reflected light fluorescence is facilitated when starting with a objective of average degreeof magnification, 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.If necessary, remove the compensator λ, which was used for the transmitted light polarizationmethod, from its compartment before adjusting the reflected light fluorescence.• Slide the fluorescence protection shield in the compensator compartment above the nosepiece.• Switch on the objective EC Plan-Neofluar 20x/0.50.• Turn the condenser turret to the position H transmitted light/bright-field first (or choose phasecontrast if using a Ph objective) and go to the sample detail to be examined.• Keep the light path in the reflected light illuminator blocked via the elimination position of the filterslider reflected light (690H690HFig. 4-16/2).