OPERATION
Carl Zeiss
Adjusting the Laser Beam with the Laser Manipulation Slider
DirectFRAP
46
423635-9001-701
05/2014
4.5
Adjusting the Laser Beam with the Laser Manipulation Slider
•
Place the specimen slide used for the adjustment, with its cover glass facing downward, on the stage
or put it into a mounting frame. If required (depending on the objective used), apply immersion oil
beforehand.
•
Switch on the HXP 120 V illuminator and move the filter set and the excitation filter required in the
manipulation experiment (see table on page 48) into position.
•
Focus on a structure in the adjustment dye of the adjustment slide using the live image in the ZEN
software (blue edition)
•
Select a diaphragm of your choice on the laser manipulation slider (Fig. 4-1/
3
).
•
Set a very low laser intensity via the ZEN software (blue edition) (preferably set 1 % via AOTF for laser
manipulation) and switch on the laser. Now, the fluorescent light of the adjustment dye should be
clearly recognizable in the live image.
•
Focus the laser manipulation slider (Fig. 4-1/
4
), i.e. focus sharply on the currently selected diaphragm.
CAUTION:
The free position (see table on page 30) is not suited for this step, since edge-sharp focusing is
not possible. If the free position is currently in the beam path, another position of the
diaphragm wheel must be selected.
•
Move now the diaphragm desired (or rather the diaphragm required for the individual experiment)
into the beam path. Check edge sharpness and refocus on the laser manipulation slider, if necessary.
From this point of time, the focal plane of the diaphragm is always located in the currently
observed image plane.
•
Position the mask centrally in the live image using the rotary buttons (Fig. 4-1/
1
and
2
).
•
Generate the software mask for the ROI position in the live image and position it exactly so that it
covers the edges of the laser beam (see the operating manual of ZEN software (blue edition)).
Repeat this procedure if the filter set or the objective are changed.
In the case of decentralized observation, the laser spot must not be shifted from the center by
more than a quarter of the camera field of view size.
Summary of Contents for DirectFRAP
Page 1: ...DirectFRAP Operating Manual...
Page 62: ......
