For TCS SP8 CARS, TCS SP8 X and Stellaris 8 FALCON/DLS

Short Instructions

1. Open FRAP Wizard from the TCS SP8/Stellaris 8 tab
2. Follow the steps in Overview tab
3. Setup imaging parameters for pre- and post-bleach imaging
   1. Only use PMT or HyD S detectors
   2. NOT HyD, HyD X or HyD R
   3. The bleaching laser intensity can damage other detectors
4. Choose Acquisition Mode (xyt or xyzt)
5. Set Acquisition Speed (fast speed is preferred)
6. Select laser lines for photobleaching
   1. On Stellaris you can use the Argon 488 nm line for more power (Drag the laser excitation to exactly 488 nm to choose it instead of the white light laser)
   2. You can select several lines if needed
   3. Use 80-100% laser power
7. Turn on the FRAP Booster for more intensity
8. Use Fly Mode to for better time resolution
9. Zoom In to the FRAP area for better photobleaching if not in Fly Mode
10. Set Background to Zero if using Fly Mode
11. Delete Bleach Images After Scan (if you’re not using the Fly Mode)
12. When you want to use different laser lines for several ROIs, uncheck Use Laser Settings For All ROIs and open ROI Configuration to choose the lasers
13. Define the number of pre-bleach, bleach and post-bleach intervals in the TimeCourse tab
    1. You can have several post-bleach steps with different number of frames and frame intervals
    2. Minimize for best time resolution
    3. Match the acquisition speed to expected recovery time; you should have at least 10 datapoints by half recovery
    4. Run the time series as long as there is increase in fluorescence intensity
14. Save your settings if you want to repeat the same experiment