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Manual
Chapter 6 –
Operating the
System
37
is the first one within each cycle. At one point in time during a certain cycle a
Click
is being applied.
The Fire will only be carried out as soon as the next cycle is being started. Thus, while the manual
Click
is unsynchronized, of course, the
Fire
initialized by the FARP controller is not.
The software "burns" a marker into the image following a "Fire" each time.
Bleach duration
. Set here the duration of the laser flash.
Acquire after fire
. With this option the following image acquisition is synchronized with the end of the
ROI scan(s). The laser spot will not be visible in this image.
Acquire while fire
. Here image acquisition and ROI scan start simultaneously. The trace of the
scanning laser spot will be visible in the image.
Multiple points
. This option allows to select several spots in the image with left mouse clicks– the
software internally generates a list of XY positions – and flash them all subsequently as fast as possible
upon a right click.
Interpolate lines
. This option is available only if option
Multiple points
is checked. Activate this option
to do "line bleaching" instead of bleaching individual spots. Apply a series of (left)
Clicks
on certain
points of a structure. The display will show a polygon line instead of spots. Start the
Fire
with a right (!)
mouse click. The scanner will move the laser beam along the line – and not switch it off between the
spots as in the usual mode.
Preview: Live view while bleaching, Live view after bleaching, Stop FRAP preview
. See previous
chapter for details.
6.3.3
Pattern Bleaching – Fluorescence Speckle Microscopy
Pattern Bleaching
command icon
In this experiment the laser spot positions are set with predefined gaps in between. The resulting
pattern of bleached or photoactivated spot makes movements of structures relative to each other quite
visible.
Most parameters and options are explained in detail in Chapter 6.3.1,
FRAP Experiments
.
Pattern spacing
Spot distance factor
. Set the value here. The
Spot size
is being multiplied with the
Spot distance
factor
In order to define a certain distance between each spot of the pattern.
Remember that the
Spot size
is defined via
Set spot size
in the
FRAP
window with the default value
presuming a perfectly focused laser; see Chapter 6.2,
The FRAP GUI in
. The pattern is most
regular if rectangular ROIs are being used. Other types of ROIs need to be composed of sub-rectangle
as described for the command
Edge accuracy
in Chapter 6.3.1,
FRAP Experiments
. Here the pattern
of spots will be more irregular.