Zeiss DirectFRAP, Operating Manual

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INSTRUMENT DESCRIPTION
Carl Zeiss Functional Principle DirectFRAP
22 423635-9001-701 05/2014
2.2 Functional Principle
The laser manipulation slider makes it possible to couple laser light simultaneously with the conventional
fluorescence illumination from the HXP 120 V light source into the reflected-light beam path of Axio
Observer. Depending on the application, the laser light is used for the targeted bleaching of fluorophores
for FRAP experiments (FRAP = Fluorescence Recovery After Photobleaching) or for the conversion of
fluorophores within the cell. The immediately following processes in the cell are observed through
fluorescence. The analysis provides detailed information on the mobility of molecules in the cell.
The optical system of the laser manipulation slider is designed in such a way that, depending on the
selected mask and magnification, a sharply limited, variously shaped ROI (Region Of Interest) in the
specimen can be illuminated with laser light.
The size and shape of the ROI can be chosen by using the diaphragm wheel in the slider. However, the
objective used has a certain influence as well (see table on page 30).
In general, the laser light is applied as a short pulse, which is controlled with high precision by the ZEN
software (blue edition) or the AOTF of the laser.
Fig. 2-1 Schematic drawing of the beam path