Zeiss Axio Imager 2 - Setting Reflected Light Fluorescence

ZEISS OPERATION Axio Imager 2
Illumination and contrast methods
192 430000-7544-001 01/2016
4.12.11 Setting reflected light fluorescence
To change the transmission, use a discrete FL attenuator (423616-0000-000 or 423617-0000-
000). The gray filters mounted in the 2-position filter wheels (428300-9901-000 or 428301-
9901-000) are not permanently stable.
The gray filter set (487935-9020-000) may be used since colored glass filters are used here.
(1) General principle
The reflected light fluorescence technique enables high-contrast images of fluorescent substances to be
displayed in typical fluorescence colors. In the reflected light fluorescence microscope, light generated by
a high-performance illuminator reaches the exciter filter (band pass) through a heat protection 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 through 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 from a spectral viewpoint. They are arranged in a
reflector module FL P&C together with the corresponding dichroic beam splitter.
(2) Instrument equipment
− Recommended objectives: EC 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 reflected light fluorescence technique, ensure that the mercury vapor short-arc
lamp is aligned by using the adjusting aid as described in Section 3.33.3
. Re-alignment may be
necessary depending on the operating time.
(3) Setting reflected light fluorescence
The initial reflected light fluorescence set up is much simpler if you begin with the EC-Plan-Neofluar
objective 20x/0.50 and a strongly fluorescing specimen. Demonstration specimens may also be used first.
Before setting reflected light fluorescence, make sure the compensator λ (Fig. 197/7
) is
removed from the slot above the nosepiece which may have been left there from
a previous
transmitted-light DIC examination.
• Switch on halogen illuminator HAL100.
• Swivel in EC-Plan-Neofluar objective 20x/0.50.
• First, swivel the condenser turret to the brightfield position H (or phase contrast Ph) and identify the
specimen feature to be examined.
• Keep the beam path in the reflected-light illuminator initially blocked with reflected-light shutter RL
(rear right on microscope stand) (indicator LED lit).