Guide for doing FRAP with Leica TCS SP2 - Biocenter

FRAP3. Identify a cell of interest and focus.Note: It can be helpful to use a fluorescent dye tocounter stain for the subcellular compartment understudy (s.a. DAPI (4',6-Diamidino-2-phenylindole) forthe nucleus). With transiently transfected cells, alsocheck that the cell is not overexpressing ypi as thiswill interfere with data analysis. Overexpressioncan disturb the endogenous protein distribution.Furthermore, this may also make it harder to definethe subcellular compartment under study.4. Adjust laser intensity, detector gain, pinhole, formatand scan speed, as well as line averaging or bidirectionalscanning if appropriate. For freely diffusing(i.e. non-binding) molecules the fastest scanspeed setting and a small image format e.g.256x256 pixels should be used. Save settings forreproducibility.Note: Make sure to set the intensity below saturationand slightly above zero as this can interfere with dataanalysis. An appropriate lookup table (glow over/under) can help. Make sure to use the same gainsettings for all cells in a given series.Initial experimentsFor a new protein one has to decide which laser linesto use for the bleaching, which bleaching intensityand length of the bleach pulse is necessary. Furthermore,depending on the size and environment of ypi,the recovery can be very rapid. Also the number ofpost-bleach images has to be optimized in order toacquire enough information for analysis and to avoidphotobleaching during post-bleach acquisition bytaking too many images.5. Now you can start the FRAP application. Doublecheckor reload the imaging settings determinedbefore. To start with, using the FlyMode is a goodidea, because it will help to find the optimum lengthfor the bleach pulse. With FlyMode you may reducethe time resolution down to 0.35 msec since the readoutof recovery is done between lines. This means,your recovery readout is closest possible to thereal zero time (t 0 ) of the postbleach intensity.The FlyMode combines both, the bleach scan andfirst image scan after bleaching. Bleaching is performedduring fly forward using ROI Scan features andhigh laser power. During fly back, the laser intensityis set to imaging values (AOTF switching withinmicro-seconds). Thus, the first image is acquiredsimultaneously with the bleaching frame. And consequently,the delay time between bleaching anddata acquisition is half the time needed to scan asingle line.Take 10 – 20 prebleach images. They determine theinitial intensity and will be important for normalizationof the data series later on. For EGFP a bleachpulse between 500 – 1000 ms with maximum laserpower often will be enough (unpublished result).This time frame may be obtained by adequate choiceof bleach iterations (i.e. if your recording interval is300 ms, setting the number of bleach iterations to 3will result in a 900 ms bleach pulse).The most importantinformation about the fluorescence recoveryis contained within the data immediately afterthe bleach pulse. Therefore the first post-bleachstep (Fig. 1) should be recorded at the highest possiblerate. For proteins which interact with otherstructures or are membrane-bound the recoveryprocess can take several minutes or even more, sofurther post-bleach steps at slower frame rates maybe necessary. Initially, to set Post 2 to 60 images at 2 sintervals and Post 3 to 30 images at 10 s intervals willcover more than 6 minutes and will be long enoughfor most diffusion processes.Note: The frame rate can be maximized by using thehighest scan speed, small image formats and bidirectionalscanning. Acquisition speed is proportionalto the number of lines scanned (y-format). The smallerthe number of pixels in x (x-format) the brighter theimage will be.Confocal Application Letter 3