PixFRET, an ImageJ plug-in for FRET calculation ... - ResearchGate

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54 J.N. FEIGE ET AL.Fig. 3. The ECFP SBT ratio can be fitted as a function of ECFPintensity. The data from Figure 1 were fitted with a linear (A) or anexponential (B) model.stant, irrespective of the gain applied to the PMT(Fig. 2B). This result suggests that the variations observedwith the set-up with two PMTs, or with the arrayof PMTs, reflect a differential response of the PMTs.Improving FRET Calculation by Modeling SBTRatios as a Function of Fluorophore IntensityTo circumvent the problem of SBT variation, using aunique PMT both for the detection of the donor and ofthe acceptor seems therefore to be the best solution.However, this imposes to acquire the three channelssequentially because of mechanical movement of thePMT, a time-consuming process that is not compatiblewith the FRET analysis of rapidly diffusing complexes.Elangovan et al. (2003) have already reported thevariation of SBT ratios as a function of fluorophoreintensity and proposed an elegant but only commerciallyavailable algorithm to tackle this problem. Alternately,we propose an easily applicable method thatconsists in modeling SBT ratios as a function of fluorophoreintensity. For the problem reported herein, theCFP SBT ratio can be fitted as a function of CFP intensityeither with a linear or an exponential model(Fig. 3). These models are then used to estimate theSBT ratio corresponding to each donor intensity andcan be implemented in the NFRET formula describedby Xia and Liu (2001) as follows:where F, CFP, and YFP are the intensities measuredwith the FRET, CFP, and YFP settings (correspond toF, D, and A in the nomenclature proposed by Gordonet al. (1998)), a and b are the average donor andacceptor SBT ratios, and c, d, e, f, and g are the constantsdetermined by the fitting of the SBT ratio,according to Figure 3.To validate this approach, the three FRET calculationmethods have then been compared with the sameset of data (Fig. 4). Cells were transfected with expressionvectors for ECFP and EYFP (negative control),ECFP fused to EYFP (positive control), or PPAR-ECFPand RXR-EYFP. The three formulas give similar resultsfor both the positive and negative FRET controls as,under these experimental conditions, variations betweencells are small and all fluorescence intensities are close tothe average of the population used to calculate SBT ratios(Fig. 4A). The linNFRET values are slightly higherbecause of the underestimation of the ECFP SBT for bothlow and high ECFP intensities (see Fig. 3A). The mostdramatic effect is seen when NFRET is calculated for theinteraction between the two nuclear receptors. Indeed,the more accurate linNFRET and expNFRET calculationssignificantly reduce standard deviation, with amore pronounced effect for expNFRET. To better characterizethe reduction of the variability of the measurementswhen modeling SBT ratios, we plotted the differencebetween expNFRET or linNFRET and NFRET as afunction of fluorophore intensity (Fig. 4B). In both cases,the highest variation was observed for extreme ECFPvalues for which using an average SBT ratio leads to agreat under- or overestimation of the correction factor(Figs. 4B and 4C). The results obtained with linNFRETand expNFRET are hence not only more precise, but alsoimprove the statistical properties of the data, allowingthe user to compare more accurately mild changes. Wealso tried to fit the donor SBT with a second order polynomialmodel rather than an exponential growth, but thismodel greatly overestimates SBT values when donorintensities are very low or very high (data not shown).Pixel-by-Pixel FRET Analysis and VisualizationCells are highly organized and protein distributionas well as interactions are often limited to specific compartments.It is hence crucial for biologists to be able tomap interactions with precision within a cell or a populationof cells to better understand the spatial organizationof cellular activities. We therefore developed anImageJ plug-in called ‘‘PixFRET’’ that allows one togenerate normalized FRET images, by computing pixelNFRET ¼ F CFPþYFP CFP CFPþYFP 3 a YFP CFPþYFP 3 bpffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiCFP CFPþYFP 3 YFP CFPþYFPð1ÞlinNFRET ¼ F CFPþYFP CFP CFPþYFP 3 ðc CFP 3 CFP CFPþYFP þ d CFP Þ YFP CFPþYFP 3 bpffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiCFP CFPþYFP 3 YFP CFPþYFPð2ÞexpNFRET ¼ F CFPþYFP CFP CFPþYFP 3 ðe CFP 3 expðCFP CFPþYFP 3 f CFP Þ þ g CFP Þ YFP CFPþYFP 3 bpffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiCFP CFPþYFP 3 YFP CFPþYFPð3Þ
PixFRET: AN IMAGEJ PLUG-IN FOR PIXEL-BY-PIXEL ANALYSIS OF FRETFig. 4. linNFRET and expNFRET reduce NFRET variability. Cos-7 cells were transfected with expression vectors for ECFP and EYFP,ECFP fused to EYFP, or PPARa-ECFP and RXRa-EYFP. Fluorescenceintensity in the ECFP, EYFP, and FRET settings was measured on atleast 50 cells. A: FRET was calculated according to the three formulasdescribed in the text. B: The difference between linNFRET orexpNFRET and NFRET calculated for the PPAR/RXR pair was plottedas a function of ECFP intensity. C: The percentage error betweenNFRET and linNFRET or expNFRET calculated for the PPAR/RXRpair was plotted as a function of ECFP intensity.by pixel the images of a sample acquired in a threechannelsetting. The plug-in allows one to either entermanually SBT parameters for the donor and theacceptor, or determine these values online using stacksof FRET and donor or acceptor images. PixFRET isfreely available on our website (http://www.unil.ch/cig/page16989.html) together with a detailed user’s guide.The PixFRET plug-in requires different stacks ofimages: (i) for FRET analysis, a stack of three imagestaken in the FRET, Donor, and Acceptor settings, (ii)for Donor SBT determination, a stack of two images ofcells expressing the Donor only and taken in the FRETand Donor settings, (iii) for Acceptor SBT determination,a stack of two images of cells expressingthe Acceptor only and taken in the FRET and Acceptorsettings.When launching PixFRET, an interface appearswhere all parameters for FRET calculation and displaycan be set (Fig. 5). The ‘‘Donor SBT’’ or‘‘Acceptor SBT’’ tabs allow one to evaluate SBTparameters (Fig. 5A). The user is first invited todraw a region outside cells to determine the backgroundin each image of the stack. Then, one or severalregions of interest on the cells have to be drawnto allow PixFRET to plot SBT ratios as a function offluorophore intensity. PixFRET then calculates themean SBT, or the parameters corresponding to thelinear or exponential fit of the data. The user candrag a sliding window on the plot to define on whichset of points the calculation is to be performed andmay then rescale the graph to the selected area byclicking the ‘‘Zoom’’ button. It is also noteworthy thatall parameters, including background (BG) values,can be directly entered manually by the user. Next,the stack with cells expressing both the donor andthe acceptor should be opened and backgroundshould be determined in the FRET tab (Fig. 5B) bydrawing a region outside cells. Backgrounds may alsobe modified manually to adjust the threshold abovewhich NFRET is calculated. Indeed, the softwareoperates NFRET calculation only when (i) the localmean pixel value, which is calculated by averagingthe values of the pixel of interest and that of its eightsurrounding neighbors, is above the mean backgroundof the image in all three images, and (ii)when the product of the mean pixel values for donorand acceptor is above the product of the backgroundsin the donor and acceptor images. These conditionswere introduced to discard points in the backgroundthat preclude the visualization of FRET within thecell by generating aberrantly high FRET valuesbecause of normalization. All background values mayalso be changed by a common multiplication factorwhich can be specified in the ‘‘Threshold CorrectionFactor’’ field. The program also offers the option tofilter the images with a Gaussian blur, the standarddeviation of the blur allowing the user to tune thesmoothing effect. Blurring notably reduces the noiseand improves the resulting image by preventingaberrant FRET values in the background. Finally,the normalization method has to be selected. Indeed,the FRET values for each pixel can be divided by thevalue in the same pixel of the donor intensity, theacceptor intensity, the product of donor and acceptorintensities, or the square root of the product of donorand acceptor intensities. Clicking on the ‘‘ComputeFRET’’ button will generate the normalized FRETimages, but also will log all parameters to a textfile which will be reloaded when the plug-in isrelaunched.FRET and NFRET are calculated only if pixel valuesin each image are above a given threshold. Otherwisethe value is set to zero in the computed imageand the pixel is displayed in blue in order to bettervisualize all the FRET values of the image (Fig. 6B).Indeed, even if within a cell the average FRET valueis positive, some pixels may have negative values,especially when the FRET efficiency is low. Since theoutput image is displayed with a 256 grayscale lookuptable, the background, which corresponds to avalue of zero, may appear in gray (and not black asintuitively expected) if negative pixels are present inthe image, thereby precluding a good visual examina-55
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PixFRET, an ImageJ plug-in for FRET calculation ... - ResearchGate

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