Zeiss Axio Observer 3 materials - Setting up Reflected Light Fluorescence Contrast

OPERATION
Axio Observer Illumination and contrast techniques ZEISS
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5.12.6 Setting up reflected light fluorescence contrast
5.12.6.1 General operating principle
The reflected light fluorescence technique enables high-contrast images of fluorescent substances to be
displayed in typical fluorescence colors. In the reflected light fluorescence microscope, light generated by
a high-performance illuminator reaches the excitation filter through a heat-absorbing filter. The filtered,
short-wave excitation light is reflected by a dichroic beam splitter and focused on the specimen via the
objective. The specimen absorbs the short-wave light and then emits the long-wave fluorescent radiation
(Stoke’s law), which is then gathered by the objective and transmitted by the dichroic beam splitter. The
light finally passes through a barrier filter which only transmits the long-wavelength light emitted by the
specimen.
Exciter and barrier filters must be perfectly matched from a spectral viewpoint. They are arranged in an FL
reflector module together with the corresponding dichroic beam splitter.
5.12.6.2 Instrument equipment
− Recommended objectives: brightfield objectives
− Reflector turret fitted with fluorescence filter set in FL P&C reflector module
or
FL filter set fitted in dual filter wheel mot. and filter wheel excitation 8-pos. mot.
− Optional: Fluorescence illuminator HXP 120 V, HBO 100, HBO 50 or Colibri 7
− microLED illuminator or HAL 100 for transmitted light illumination
− Recommended: Switching mirror for 2 illuminators and a further iris diaphragm slider for slot A.
Before using the reflected light fluorescence technique, ensure that the mercury vapor short-arc
lamp is aligned as described in section 4.23.3
. Re-
alignment may be necessary depending on
the operating time. The self-adjusting HBO 100 can also be used.
5.12.6.3 Setting reflected light fluorescence
The initial reflected light fluorescence set up is much simpler if you begin with the Plan Neofluar 20x/0.5
objective or EC Plan Neofluar 20x/0.5 objective and a strongly fluorescing specimen. Demonstration
specimens may also be used.
• First search for the specimen in the reflected light brightfield (see section 5.12.5) or transmitted light
brightfield (see section 5.12.1).
• Switch the light path from the microLED or HAL 100 illuminator in transmitted light to HXP 120 V or
HBO illuminator in reflected light.
• If in use, replace the aperture diaphragm slider MAT by the iris diaphragm slider.
• First use the internal fluorescence shutter to keep the light path in the reflected light section closed by
pressing the RL button (Fig. 154/10).