Guide for doing FRAP with Leica TCS SP2 - Biocenter

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FRAPFigure 5 (raw, Pre) shows a prebleach image of amouse fibroblast nucleus transfected with histone H1fused to GFP with bleach ROI (green), whole cell ROI(red) and the unbleached part of the cell (orange)overlaid. Figure 5 also shows the first postbleach(Post1) and the last image (Post3) of a FRAP serieswith the same cell.Data processingAs mentioned above the values for the characteristicrelaxation time τ, the recovery half-time t 1/2 and therecovery rate are only rough estimates, because thedata has not been corrected. There are three necessarycorrections: Background subtraction, correctionfor (unintended) photobleaching during acquisition ofpost-bleach images and finally normalization (Figure 2).All three will be explained in the following.Figure 3 illustrates the meaning and positions of thedifferent regions of interest (ROIs) used. For clarityROIs are not drawn to scale and the bleach ROI isdepicted in red, but will be depicted in green with actualdata (see below).Background subtractionAll images contain a background signal. Some of it isdue to noise of the photomultiplier tubes (PMTs) andthe electronics, some is background fluorescencedue to residual staining or overexpression or mislocationof the GFP-tagged protein. The former kind ofbackground originates from the hardware and canonly be reduced by setting a lower PMT voltage at theexpense of sensitivity. It can be treated by recordingan image with all lasers turned off and the respectivePMT turned on. This “noise image” can later on besubtracted from the image series.Note: Make sure that there are no pixels with zerointensity. You can check for zero values with the“O/U LUT (glow over/under)” (they are green). If thereare zero values increase the offset of the PMT. Thehistogram can also help to find the backgroundvalue. The simulation software by Siggia et al. (2000)uses the histogram peak near the low end of thehistogram for background subtraction.If the image contains parts of a cell which is not stainedin the respective channel, background due tofluorescence can be corrected for by subtracting theaveraged intensity of unstained parts from the FRAPseries. In the example Figure 5 (upper row, right) theaveraged intensity of ROI 4 (cyan) would be subtracted.Figure 2Necessary corrections represented schematically. Raw data givenin dark red (dots), after background subtraction (red dash-dots) andcorrection for photobleaching artifacts (solid blue). Note the intensitydecrease after time 100 caused by photobleaching during acquisition.After normalization relative fluorescence values are plotted over time(right figure, solid black line).Correct for fluorescence lossdue to photobleachingWe have to distinguish two kinds of photobleaching:Firstly, the intended photobleach with full laser intensityduring the bleach pulse, secondly, photobleachingduring post-bleach acquisition. While the formerphotobleaching is intended, the latter is inherent tothe imaging process and can lead to some considerableloss of total fluorescence. Therefore, the fluorescenceintensity can never recover to 100%, even if allfluorophore molecules were mobile (see “Calculationof mobile fraction”). It is possible to correct for thisamount of photobleaching by multiplying every element6Confocal Application Letter
FRAPNote: Alternatively, a second cell, which does nottake part in the FRAP experiment, can be used as anindependent control. In this case the ratio F precell /F infcell would be determined for this cell.Note: F precell /F infcell can only be accurately determinedfor a closed compartment without exchangewith the surroundings within the time-scale of theexperiment (s.a. the nucleus in our live cell example).In spite of this limitation the data from the FITCdextranexperiment was used here, for illustrationpurposes only.NormalizationNormalization means to rescale the images in a waythat represents plateau fluorescence as 100% (fractionalfluoresence intensity) in order to make severalimage series mutually comparable as well as to enableus to readily estimate the relaxation half-time t 1/2 .The most commonly employed normalization usedwas introduced by Siggia et al. (2000). Here everyframe (time point) is divided by the first frame (timepoint). This way Pre-Bleach intensities will be normalizedto 100%. This type of normalization is suitable tographically determine the mobile fraction from normalizeddata, while the relaxation half-time can not.This method can also be applied to incomplete recoveries(i.e. fluorescence intensity has not reached aplateau value.).Figure 3Schematic cell expressing GFP mainly in the nucleus. A region is bleachedin the nucleus (red circle). After the bleach pulse the cell isimaged at low intesity to follow the fluorescence recovery. Pre-Bleach shown with Bleach ROI (region of interest) as well as furtherquantification ROIs (not drawn to scale).of the data set by (F precell /F infcell ), where F precell meansprebleach intensity within the whole cell, F infcellstands for postbleach intensity (whole cell minusBleach ROI) at any given time point. This correctionshould be done after subtraction (see above). In anycase, it is preferable to optimize the experimentalparameters in order to avoid strong photobleaching.An acceptable range would be between 5-15% fluorescenceloss.An alternative method was published by Axelrod et al.(1976).Fn(t) = (F(t) – F(0))/(F(inf) – F(0)) (1)WithF(t)..fluorescence intensity at time t inside bleach ROIF(0)..fluorescence intensity at time 0(immediately after bleaching)F(inf)..fluorescence intensity at equilibriumUsing the “Axelrod method” the plateau value is setto 100%. Therefore, the relaxation half-time can begraphically determined from a normalized plot, whilethe mobile fraction cannot be determined.The following sections introduce methods for formalestimation of mobile fraction and relaxation half-times.Confocal Application Letter 7
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