Frap/Bleaching Experiments - Nikon A1+ User Manual

FRAP/Bleaching Experiments

1. This section of the manual describes how to enable and use the bleaching features
of the confocal and only serves as a reference if you have already been trained to
perform FRAP experiments by facility staff
2. Select the FRAP-FRET layout from the bottom left corner of the screen. This will
show you additional toolbars required to carry
out a FRAP experiment
3. A FRAP acquisition usually consists of several phases which are run sequentially:
Acquisition-A few frames are acquired of the cell before bleaching
Bleaching-A number of scans are acquired at high laser power of an ROI containing the
area to be bleached
Acquisition-Many frames are acquired post bleaching in order to monitor the fluorescence
recovery within the bleached area
4. Use the ND Stimulation window to configure
the required phases. Select the tick box
under the phase column to add a phase
5. Under the Acq/Stim heading you can select
what procedure happens during that
particular phase i.e. acquisition or bleaching
6. The Interval, Duration and Loops columns
are used to configure over what time period
that particular phase runs
7. So for an Acquisition phase the images will
be acquired with the capture settings you
have configured in the A1Plus Compact GUI
8. Tick the Perform Time Measurement box so that the intensity values within the
ROI(s) are monitored during the entire experiment.
9. The capture settings used during the bleaching phase are configured using the
A1plus Stimulation window
10. Select the Synchronize lasers tick box if you'd like the same
laser lines/power to be used on all image bleaching ROIs
11. HV Mode should be set to "Keep HV". This feature selects
whether the detector gain is set to zero during the non-
acquisition bleaching phase to avoid detector overload which
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