Zeiss DirectFRAP, Operating Manual

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OPERATION
DirectFRAP Operation via ZEN Software (Blue Edition) Carl Zeiss
05/2014 423635-9001-701 47
4.6 Operation via ZEN Software (Blue Edition)
The laser manipulation is controlled via the ZEN software (blue edition).
For operating the software please see the ZEN operating manual (blue edition).
DirectFRAP requires the fast acquisition module. The physiology module is recommended in addition. The
planning and recording of the experiment is done using the software.
4.7 Experiment Procedure
•
Put the cultivation vessel, containing the specimen to be examined, on the stage or insert it into the
mounting frame.
•
Select a cell for adjusting the laser energy and focus it using the configuration for cell localization (see
table on the next page).
•
Activate the ROI software mask and position the cell with the help of the stage drive in such a way
that the ROI mask marks the region desired for manipulation.
•
Select now the filter combination for the manipulation process (see table on the next page).
•
Trigger laser pulses via the ZEN software (blue edition) and evaluate the bleaching or conversion
efficiency, respectively, by comparing the mean image gray scale values of the ROI beforehand and
afterwards. Increase the laser energy slowly until the pulse duration for the desired manipulation
efficiency has decreased to about 0.2 seconds (or less, depending on the application).
•
Finally, find another cell for the actual experiment and carry out the manipulation process using the
laser energy determined.
To avoid undesired conversions caused by short-wave light emitted by the HXP 120 V
illuminator, all positions of the excitation filter wheel not used must be provided with blocking
disks. If other experiments require the use of short-wave-length transmitting excitation filters
incorporated in the filter wheel, the reflected-light shutter of Axio Observer has to be closed
before the filter wheel is switched to another position.