Setting Of Epi-Fluorescence - Zeiss Axioskop 2 plus Operating Manual

Axioskop 2 plus
Axioskop 2 mot plus
3.3.6

Setting of epi-fluorescence

(1) General principle
The epi-fluorescence technique permits high contrast images of fluorescent substances in typical
fluorescence colors. In the epi-fluorescence microscope, light generated by a high-performance
illuminator reaches the excitation filter (band-pass) via a heat protection filter. The filtered, short-wave
excitation emission is reflected by a dichroic beam splitter and focused on the specimen via the objective.
The specimen absorbs the short-wave emission and then emits the long-wave fluorescence (Stoke's law),
which is now gathered by the objective and transmitted by the dichroic beam splitter. Finally, the rays
pass a barrier filter (long pass/band pass), which only allows the long-wave emission from the specimen
to be transmitted.
Excitation and barrier filters, which are both positioned in the FL P&C reflector module together with the
relevant dichroic beam splitter, must be perfectly matched.
(2) Axioskop 2 plus and Axioskop 2 mot plus configuration
− Recommended objectives: Plan-Neofluar or Fluar (UV-excitation)
− FL P&C reflector module and shutter plate in the reflector turret
− HBO 103 or HBO 50 mercury vapor short-arc lamp for incident-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 section 2.1.11 by using the adjusting aid. If required,
re-adjustment must be performed depending on the operation time.
(3) Setting of epi-fluorescence on the Axioskop 2 plus and Axioskop 2 mot plus
The first epi-fluorescence setting is considerably facilitated if the Plan-Neofluar objective 20x/0.50 and a
specimen featuring pronounced fluorescence is used. It is also possible to use demonstration specimens
first.
☞
If the compensator λ (3-26/6) has not been removed from the compartment above the
nosepiece after transmitted-light DIC microscopy, it must be taken out before setting epi-
fluorescence.
• Switch on the HAL100 halogen illuminator.
• Swing in Plan-Neofluar 20x/0.50 objective.
• Move condenser turret to position H, transmitted-light brightfield (or also phase contrast), and then
move to the specimen area to be examined.
• Keep light path in the incident-light illuminator blocked at first using shutter plate on the reflector
turret (3-27/1) or the barrier position of the incident-light filter slider (3-27/5).
B 40-075 e 02/01
OPERATION
Illumination and contrasting techniques Carl Zeiss
3-39