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