FRAP Group 3a Alessandro Borgia Juliane Winkler

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Presentation transcript:

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

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

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

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

Increasing the ROI 1 4

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

Cytosolic GFP

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).

THANKS TO: Stefan Terjung Christian Tischer YOU ALL