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 (
4-120/7) 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).
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