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FRAP Measure the intracellular (HeLa) diffusion of: EGF (membrane bound) GFP in cytosol

Group 3a Alessandro Borgia Juliane Winkler. FRAP Measure the intracellular (HeLa) diffusion of: EGF (membrane bound) GFP in cytosol. FRAP: membrane bound EGF-receptor. TIME (AFTER BLEACHING). t=100 ms. t=1000 ms. t=1500 ms. t=3000 ms. t=0. FRAP: membrane bound EGF receptor. ROI.

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FRAP Measure the intracellular (HeLa) diffusion of: EGF (membrane bound) GFP in cytosol

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

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

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

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

  5. Increasing the ROI 1 4

  6. Cytosolic GFP 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

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