OPERATIONCarl Zeiss Illumination and Contrast Techniques in Reflected Light Axio Vert.A194 431030-7044-001 05/20124.12.5 Reflected Light FluorescenceThe reflected light fluorescence method allows fluorescent substances to be presented in a high-contrastmanner and in typical fluorescence colors. In a reflected light fluorescence microscope, the light producedby a high-performance illuminator reaches the excitation filter through a heat protection filter. Thefiltered short-wavelength excitation radiation is reflected by a dichroic beam splitter and then focused onthe specimen via the objective. The specimen absorbs the short-wavelength radiation and subsequentlyemits fluorescence radiation of a longer wavelength (Stoke's Law), which is picked up as an image by theobjective and transmitted by the dichroic beam splitter. The rays eventually pass through a barrier filter,which allows only the long-wavelength radiation emitted by the specimen to go through.The excitation and barrier filters need to be very accurately matched spectrally and are located in an FLP&C reflector module, together with the corresponding dichroic beam splitter.Requirements− Stand Axio Vert.A1 FL or Axio Vert.A1 with HBO / HXP illuminatoror− Axio Vert.A1 FL-LED stand with LED modules mounted− The microscope must have been properly put into operation and switched on, as described inSection 3.− Bright field objectives− FL P&C reflector module in the reflector turret− Illuminators for fluorescence (HBO 50, HBO 100, Colibri 2, HXP 120 C)− Prior to their use, the HBO 50 or HBO 100 illuminators need to be adjusted and warmed up for about15 minutes, as described in Section 3.1.11.2 and 3.1.11.3, respectively.− HAL illuminator or LED illuminator for transmitted light illuminationSetting Fluorescence• Select the area of the specimen in transmitted light bright field or phase contrast. To this end, switchthe reflector turret (Fig. 4-17/4) to free passage and set the position for bright field or phase contraston the condenser (Fig. 4-17/2).• Use the power supply unit (Fig. 4-17/7) to turn on the fluorescence illumination, e.g., the HBO 50illuminator (Fig. 4-17/3).• Use the reflected light shutter (Fig. 2-7/25) to keep the light path temporarily blocked.• Once you have selected the area of the specimen, use the on/off function (Fig. 4-17/5 and 6) toswitch off the transmitted light illumination (Fig. 4-17/1).When you employ the Axio Vert.A1 FL-LED stand, the TL/RL switch (Fig. 2-8/20) lets you shiftdirectly between transmitted light and reflected light.• Using the reflector turret (Fig. 4-17/4), rotate in the desired filter set and unblock the light path withthe reflected light shutter (Fig. 2-7/25).• Close the luminous field diaphragm (Fig. 4-17/10) until it is visible in the image. Using the centeringscrews, move the diaphragm into the center position and open it all the way to the edge of the fieldof view.
