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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