Axioskop 2
plus
OPERATION
Axioskop 2
mot plus
Illumination and contrasting techniques
Carl Zeiss
B 40-075 e 02/01
3-39
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
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/
) or the barrier position of the incident-light filter slider (3-27/
5
).
