Setting Of Fluorescence Contrast In Reflected Light - Zeiss Axiovert 200 Operating Manual

Carl Zeiss
3.3.5

Setting of fluorescence contrast in reflected light

3.3.5.1 General principle
The epi-fluorescence technique permits high-contrast images of fluorescent substances in typical
fluorescence colors. In the epi-fluorescence microscope, the light generated by a high-performance
illuminator reaches the excitation filter via a heat-reflecting filter. The filtered, short-wave excitation
emission is reflected from a dichroic beam splitter and is focused on the specimen via the objective. The
specimen absorbs the short-wave emission and then emits long wave fluorescence (STOKE's law) which
is now gathered by the objective and transmitted by the dichroic beam splitter. Finally, the beams pass a
barrier filter which only allows the long-wave emission from the specimen to be transmitted.
Excitation and barrier filters, which are positioned in the FL reflector module together with the
appropriate dichroic beam splitter, must be perfectly matched.
3.3.5.2 Configuration of the Axiovert 200 (manual) and Axiovert 200 M
Recommended objectives: brightfield objectives
FL reflector module in the reflector turret
N HBO 103 fluorescence illuminator or HBO 50 for reflected-light illumination
HAL 100 halogen illuminator for transmitted-light illumination
Before the epi-fluorescence technique is applied, it is absolutely necessary to adjust the
mercury vapor short-arc lamp in accordance with sections 2.12.2 through 2.12.4 by using the
adjusting aid. If required, re-adjustment must be performed depending on the operation time.
3.3.5.3 Setting of epi-fluorescence on the Axiovert 200 (manual) and Axiovert 200 M
The first epi-fluorescence setting is considerably facilitated if the Plan-Neofluar objective 20x/0.50 and a
specimen featuring pronounced fluorescence are used. It is also possible to use demonstration specimens
first.
Switch on the HAL 100.
Swing in suitable objective, e.g. Plan-Neofluar 20x/0.50, via the nosepiece (3-24/4).
Move condenser turret to position H, transmitted-light brightfield (or also phase contrast), and then
move to the specimen area to be examined.
Use focusing drive for focusing.
Keep light path in the reflected-light part blocked at first using the fluorescence shutter by pressing
the FL on / off key (3-24/6).
3-40
OPERATION
Illumination and contrasting techniques Axiovert 200
B 40-080 e 03/01