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OPERATION
Carl Zeiss
Illumination and contrast methods
Axio Imager
176
430000-7344-001
M70-2-0020 e 06/2009
4.9.11
Setting epi-fluorescence
CAUTION
To reduce the transmission, use an FL attenuator, discrete (423616-0000-000 or 423617-
0000-000). The gray filters mounted in the 2-position filter wheels (428300-0000-000 or
428301-0000-000) are not permanently stable.
(1) General
principle
The epi-fluorescence technique enables high-contrast images of fluorescent substances to be displayed in
typical fluorescence colors. In the epi-fluorescence microscope, light generated by a high-performance
illuminator reaches the exciter filter (band pass) 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 fluorescence light (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 light from the specimen to be
transmitted.
Exciter and barrier filters must be perfectly matched. They are arranged in a reflector module FL P&C
together with the corresponding dichroic beam splitter.
(2) Instrument
configuration
−
Recommended objectives: Plan-Neofluar or Fluar objectives (UV excitation)
−
Reflector module FL P&C in reflector turret
−
Mercury vapor short-arc lamp HBO 100 for reflected-light illumination
−
Halogen illuminator HAL 100 for transmitted-light illumination
Before using the epi-fluorescence technique, make sure to align the mercury vapor short-arc
lamp by means of the adjusting aid as described in Section 3.31.3. Re-alignment may be
necessary depending on the operating time.
(3) Setting
epi-fluorescence
The first epi-fluorescence setting is considerably simplified if you begin with the Plan-Neofluar objective
20x/0.50 and a strongly fluorescing specimen. You may also use demonstration specimens first.
Before setting epi-fluorescence, make sure to remove compensator
λ
7
) from the slot
above the nosepiece, which may have been left there from a previously performed transmitted-
light DIC examination.
•
Switch on halogen illuminator HAL100.
•
Swivel in Plan-Neofluar objective 20x/0.50.
•
First, swivel condenser turret to brightfield position H (or phase contrast Ph) and set the specimen
feature to be examined.
•
For the time being, keep the light path in the reflected-light illuminator blocked by reflected-light
shutter RL (rear right on microscope stand) (indicator LED is lighting).