1. FRAP Group 3a Alessandro Borgia Juliane Winkler
Measure the intracellular (HeLa) diffusion of:
EGF (membrane bound)
GFP in cytosol

2. FRAP: membrane bound EGF-receptor
TIME (AFTER BLEACHING)
t=0
t=100 ms
t=1000 ms
t=1500 ms
t=3000 ms

3. FRAP: membrane bound EGF receptor
whole cell intensity (cell)
mean fluorescence intensity
ROI
Background (BG)
Time (sec)

4. mean fluorescence intensity
EGF-receptor
cell
ROI
background
the average intensity of the prebleach image is set to 1
Time (sec)
mean fluorescence intensity
FLUORESCENCE
RECOVERY
CURVE

5. Increasing the ROI 1 4

6. Cytosolic GFP TIME (AFTER BLEACHING) = 130 ms TIME (AB) = 195 ms
PREBLEACH
TIME (AFTER BLEACHING) = 130 ms
TIME (AB) = 195 ms
TIME (AB) = 455 ms

7. Cytosolic GFP

8. TROUBLESHOOTING (or troubles shooting at us, you decide...)
1ST BLEACHING - SIGNAL POOR  ENOUGH LASER POWER  ADJUSTED TO MAX
DATA STILL VERY NOISY - HOW TO IMPROVE IT?
HIGHTEN PHOTOMOLTIPLICATION OR GAIN
INCREASE SPOT AREA  INCREASES OVERALL SIGNAL INTENSITY
BUT IT TAKES LONGER TO FULLY RECOVER THE FLUO  LONGER EXPOSURE TO IMAGING LASER
HIGHER PHOTOBLEACHING OF THE WHOLE CELL FROM IMAGING LASER  DID NOT WORK
OPTIMIZATION OF THE ACQUISITION ALTOGETHER:
REDUCE SCANNING AREA („CLIPPING“)
ADJUST BLEACHING TIME TO TRIGGER THE BLEACHING WHEN THE SCANNER IS VERY CLOSE TO THE BLEACHED SPOT
INCREASE PIXEL SIZE AND TRIED TO SCAN BIDIRECTIONALLY - LOSE RESOLUTION!
FOR CYTOSOL ONLY  OPEN THE PINHOLE FULLY - INCREASES SIGNAL AND IS LESS SENSITIVE TO MOTION ALONG Z-AXIS (NO DEFOCUSING PROBLEMS).

9. THANKS TO:
Stefan Terjung
Christian Tischer
YOU ALL