Guide for doing FRAP with Leica TCS SP2 - Biocenter

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FRAPFigure 1Live cell: Fluorescence recoveryplotted versus time in 8-bitformat (i.e. max = 255). Timeintegrated grey values (intensities)of bleach ROI (green),whole cell ROI (purple), unbleachedpart of cell (orange,more information, see text).Three iterations with bleachintensity were applied (betweenarrows). The backgroundintensity was not takeninto consideration in this example. Note: Only in the Fly-Mode you are able to monitorthe fluorescence decay duringbleaching.6. Press next. Now the laser intensity for bleachinghas to be set. EGFP or FITC have an absorbancemaximum near the 488 nm line of an Ar-Ion laser.Additionally activating the 458 and 476 nm laserlines can increase the bleaching depth. The bleachingdepth is the ratio between the post-bleach intensityF 0 and the prebleach intensity within thebleached region. F 0 should be close or equal tobackground intensity, otherwise the curvature ofthe recovery curve will be small which can bedetrimental to quantitative data analysis (see below).Note: It can be difficult to bleach rapidly diffusingspecies to background intensity. In this case establishingthe FRAP procedure using a fixed sample canhelp (Snapp et al. 2003).Note: There are different geometries for bleach ROIsavailable in LCS. The most widely used ones are spotphotobleaching (circular ROI) and strip photobleaching(rectangular ROI extending beyond the bordersof the labelled organelle). Strip photobleaching hasbeen described by Snapp et al. (2003), Ellenberg etal. (1997) and Siggia et al. (2000), while spot photobleachingwas used in the landmark paper by Axelrodet al. (1976) and for FRAP experiments in the nucleus(Phair and Misteli, 2000).8. Run experiment. After the experiment has finished,a window will open plotting the integrated intensityfor each ROI (Figure 1, Figure 5, raw). The softwareoffers a simple exponential fit to the FRAP datagiving an estimate of the time constant, characteristichalf time and rate of the recovery. However thisfit result has to be considered preliminary, becauseat this point no background subtraction, bleachingcorrection and normalization has been carried out.For data processing export the ROI data as a text file.You can then import it into your favourite data processingor statistics software (i.e. Excel, Mathematica,Matlab or others).Note: In order to be able to repeat the experimentwith exactly the same settings (i.e. for repeated photobleachingof the same cells as a test for phototoxicityand reproducibility) it is recommended to save thesettings.7. Define a region of interest (ROI) for bleaching. It isalways a good idea to save the ROI for quantificationlater on (right-click in the image window/ROI/save).Note: The larger the ROI the longer the recovery willtake. Proportionally, the total fluorescence intensityof the whole cell is reduced which aggravates theproblem of photobleaching during post-bleachacquisition. As a rule of thumb use a larger ROI forrapidly diffusing proteins and a smaller one for moreslowly diffusing proteins.9. Repeat the FRAP experiment with other cells in thesample (about a dozen cells for some basic statistics).FRAP experimentsTo provide an example demonstrating the FRAP procedurewe will use a 464 kD dextran labelled withfluorescein-isothiocyanate (FITC-dextran, FD464), whichis commercially available from most laboratory suppliers.The dextrans can serve as a system to practicethe steps of a real FRAP experiment without having touse live cells. A FRAP experiment using live cells willalso be given.4Confocal Application Letter
FRAPPhotobleaching a fluorescein-labelledpolymer in solutionFITC-dextran (Sigma-Aldrich) of MW 464,000 wasprepared in PBS buffer pH 7.4 at 1.5 mg/ml with 30%(w/w) glucose added. The solution was filled intomicroslides with ground cavity (Karl Hecht KG, Sondheim,Germany), covered with a coplanar coverslipand sealed with acetone-free nail polish.Experimental conditions were:Ojective63x NA 1.4 OilScan modexytScan speed1400 HzExcitation wavelength488 nmEmission range498-600 nmFormat 256 x 256Zoom 8Laser power/potentiometer (Ar/KrAr) ~75 %AOTF (imaging) 488 nm 1.5 %AOTF (bleaching) 488 nm 100%ROI geometryCircleROI diameter 9.2 µmPrebleach20 x 294 msBleach3 x 294 msPost 115 x 294 msPost 260 x 1 sPost 350 x 5 sNote: One can be tricked into believing the plateau isreached at this point, because photobleaching duringpost-bleach acquisition decreases the signal andcan mimic an equilibrium state (this will becomeapparent after bleaching correction during data analysis).See data processing.As useful as the FlyMode is, sometimes an even higherbleach depth is needed. For this purpose one can activatethe Zoom-In option (cannot be combined with FlyMode).With this option checked, the aspect ratio of the imagewill be adjusted to accommodate the bleach ROI resultingin a net zoom in during the bleach. As a resultevery pixel will be irradiated more often during the scanwhich leads to a larger fluorescence loss.Note: During the bleach with ZoomIn no images willbe recorded, so the recovery curve will have a ‘hole’(e.g. will not be steady).Note: If really maximum recording speed is essential,the time lag between the acquisition of two subsequentimages can be minimized by switching on“complete burst” mode. With this setting image acquisitionis at maximum speed. Therefore the screenwill not show any live images during recording.After having established experimental conditions wenow have to optimize the bleach pulse as well as thenumber and rate of post-bleach images. For this purposethe FlyMode is activated. It allows us to examinethe bleaching process in detail. For the first trial ableach pulse of 4 x 294 ms = 1,2 s was set. Figure 1shows the first 40 Post-bleach images. From the plot itbecomes evident that the last bleach iteration onlyresults in a ~5% decrease of fluorescence intensity.Therefore, for the following experiments only 3 iterationswill be used. This way we can optimize the lengthof the bleach pulse and can now monitor the early onsetof the fluorescence recovery.In much the same way the graphical view helps us tooptimize Post 1 step. After 15 iterations most of therecovery has taken place, so that for Post 2 and 3 aslower rate can be used. If the intensity has notaltered for about 1 min while observing the recovery,it can be assumed to have reached its asymptotic(plateau) value.FRAP of a nuclear protein in live cellsHistone H1 – tagged with EGFP (contruct kindly providedby T. Misteli) was transfected into 3T3 NIH mousefibroblast cells. A FRAP experiment was conductedwith the following conditions:Experimental conditions were:Ojective63x NA 1.4 OilScan modexytScan speed1400 HzExcitation wavelength488 nmEmission range498-600 nmFormat256x256Zoom ~ 8Laser power/potentiometer (Ar/KrAr) ~75 %AOTF (imaging) 488 nm 1.2 %AOTF (bleaching) 488 nm 100 %AOTF (bleaching) 476 nm 100 %AOTF (bleaching) 456 nm 100 %ROI geometryPolygonROI size (enclosing rectangle) 15.3 µm x 4.1 µmPrebleach20 x 294 msBleach3 x 294 msPost 140 x 294 msPost 230 x 2 sPost 324 x 10 sConfocal Application Letter 5
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Guide for doing FRAP with Leica TCS SP2 - Biocenter

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