frap measure the intracellular (hela) diffusion of: egf (membrane bound) gfp in cytosol
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
FRAP
Measure the intracellular (HeLa) diffusion of:
EGF (membrane bound)
GFP in cytosol
Group 3a
Alessandro Borgia
Juliane Winkler
t=0
FRAP: membrane bound EGF-receptor
t=100 ms t=1000 ms t=1500 ms t=3000 ms
TIME (AFTER BLEACHING)
FRAP: membrane bound EGF receptor
Background (BG)
ROI
whole cell intensity (cell)
Time (sec)
mea
n flu
ores
cenc
e in
tens
ity
EGF-receptorcell
ROI
background
Time (sec)
mea
n flu
ores
cenc
e in
tens
ity
FLUORESCENCERECOVERY
CURVE
€
DATA NORMALIZATION =f ROI t( ) − f BG t( )
f WHOLE CELL t( ) − f BG t( )
⎛
⎝ ⎜ ⎜
⎞
⎠ ⎟ ⎟− offset
the average intensity of the prebleach image is set to 1
Increasing the ROI
1 4
TIME (AFTER BLEACHING) = 130 ms
TIME (AB) = 195 ms
TIME (AB) = 455 ms
PREBLEACH
Cytosolic GFP
Cytosolic GFP
1ST BLEACHING - SIGNAL POOR ENOUGH LASER POWER ADJUSTED TO MAX
TROUBLESHOOTING (or troubles shooting at us, you decide...)
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