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

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52 J.N. FEIGE ET AL.thereafter show that a simple modeling to estimatethese SBT ratios as a function of intensity greatlyimproves the accuracy of FRET calculation.Another important interest for biologists when analyzingFRET in living cells concerns the location of theinteraction studied within the cell or the cell population.We report here the development of an ImageJplug-in, called ‘‘PixFRET,’’ which allows one to calculateand display FRET directly on images by performinga pixel-by-pixel analysis of images and assisting inthe determination of SBTs.The usefulness of the modeling of SBT ratios and ofthe pixFRET plug-in is exemplified using the enhancedcyan and yellow fluorescent proteins (ECFP andEYFP), the FRET pair which is today the most widelyused by biologists to study interactions of proteins inliving cells, fused to each partner of a well-establishedheterodimer: the peroxisome proliferator-activatedreceptor (PPAR) and the retinoid X receptor (RXR)(Feige et al., 2005).MATERIALS AND METHODSPlasmid Constructs and ReagentscDNAs encoding mouse PPARa as well as RXRa weresubcloned after PCR amplification into the pEYFP-N1and pECFP-N1 plasmids (BD Biosciences Clontech,Switzerland) as described previously (Feige et al., 2005).The ECFP–DEVD–EYFP construct and GI262570-FITCwere kind gifts of Dr. J.M. Tavaré (Rehm et al., 2002)and Dr. Peterson (DeGrazia et al., 2003), respectively.Cell Culture and Transient Transfection AssaysCos-7 cells were maintained in Dulbecco’s modifiedEagle’s medium supplemented with 10% fetal calfserum (Gibco/Invitrogen, Switzerland). Penicillin andstreptomycin (Gibco) were added to the media at 100units/ml and 100 lg/ml, respectively.Transient transfection assays were performed usingLipofectamine 2000 (Invitrogen, Switzerland). Cellswere plated in 4-well LabTek chambered coverglasses(Nunc) for microscopy studies.Confocal ImagingLive cells grown on LabTek chambered coverglasseswere washed once with phenol red free Optimemmedium (Gibco) and observed in the same medium.Observations were performed at 378C on a TCS SP2AOBS confocal microscope (Leica, Germany) equippedwith four photomultipliers (PMTs) and with a wholemicroscopeincubator (Life Imaging Service, Switzerland).Additional experiments were performed on anLSM510 Meta confocal microscope, using the Metaarray of PMTs (Zeiss, Germany), and on an OlympusIX70 wide-field microscope equipped with a PolychromeII monochromator (Photonics, USA) set at420 nm, a Zeiss 488027 filter set (Ex BP 410/16 þ 489/22 – Em BP 456/17 þ 535/44) or an Olympus YFP filterset (BP535/30) and an Imago charge-coupled device(CCD) camera (Photonics, USA). Acquisitions were performedwith a 63X/NA 1.2 water or a 63X/NA 1.4 oilimmersion objective. Quantification of images was performedusing either the Leica Confocal Software (LCS)version 2.4 or ImageJ version 1.33.For FRET experiments, transfections were performedas described above and expression levels ofdonor and acceptor proteins were adjusted to similarlevels by Western blot. Unless otherwise stated, thethree different settings used for the analysis of FRETwith the CFP/YFP pair were (i) FRET: Ex 405 nm/Em525–545 nm, (ii) CFP: Ex 405 nm/Em 465–485 nm, (iii)YFP: Ex 514 nm/Em 525–545 nm. Laser power anddetector gain were adjusted in the different channelsin order to observe equimolar concentrations of CFPand YFP at equal intensities (equimolar concentrationsof CFP and YFP were obtained by expressing a referencefusion protein of CFP and YFP spaced by 475 residues).The analysis of FRET with the FITC/Cy3 pairwas performed with the following settings: (i) FRET:Ex 488 nm/Em 565–585 nm, (ii) FITC: Ex 488 nm/Em510–530 nm, (iii) Cy3: Ex 514 nm/Em 565–585 nm. Settingswere kept unchanged for analysis of all samples.Unless otherwise stated, donor emission was detectedon the photomultiplier 1 (PMT1) and acceptor andFRET emissions were recorded on PMT2. Donor andacceptor SBTs in the FRET setting were determined oncells expressing the donor or the acceptor alone bycalculating the intensity (I) ratios in the appropriatesettings (I FRET /I Donor and I FRET /I Acceptor , respectively).Linear and exponential fits were performed usingMicrosoft Excel and FindGraph, respectively. FRETmeasured in coexpressing cells was then corrected forSBTs and normalized (NFRET) for expression levelsaccording to the following formula (Xia and Liu, 2001):NFRET ¼ I FRET I Donor 3 BT Donor I Acceptor 3 BT AcceptorpffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiI Donor 3 I AcceptorProgram for the Plug-inThe program PixFRET presented in this paper is aplug-in of the public-domain software ImageJ. ImageJis a general-purpose image-processing program; it isthe Java offspring of the NIH Image software. As aresult, it can run on any platform with a Java VirtualMachine (Macintosh, Windows, Unix, etc.). The applicationand its source are available at http://rsb.info.nih.gov/ij/. PixFRET is freely available at http://www.unil.ch/cig/page16989.html.RESULTSSBT Ratios Can Vary as a Functionof Fluorophore IntensityTo study FRET in living cells, we chose two nuclearreceptors previously shown to form heterodimers invitro and in vivo, PPAR and RXR that were, respectively,fused to ECFP (donor) and EYFP (acceptor)(Feige et al., 2005).An important parameter in FRET experiments is theamount of SBT between channels. To estimate SBTs,average fluorescence intensities in cells expressingonly the donor (or the acceptor) were quantified, bothin the FRET and in the donor (or acceptor) channels.The ratios between the fluorescence intensities in eachchannel were calculated after background subtraction.In cells expressing PPAR-CFP alone, the donor SBTratio, defined as the ratio between the amount of light
PixFRET: AN IMAGEJ PLUG-IN FOR PIXEL-BY-PIXEL ANALYSIS OF FRET53Fig. 1. The donor bleed-through ratio increases with donor intensity.A: Cos-7 cells were transfected with an expression vector forPPARa-ECFP. Fluorescence intensity in the ECFP (Exc. 405 nm/Em.465–485 nm, PMT1) and FRET (Exc. 405 nm/Em. 525–545 nm,PMT2) settings was measured on at least 300 cells. The SBT ratio isthe ratio between the average FRET and ECFP intensities measuredin individual cells. F CFP and CFP CFP are the intensities measured inthe FRET and CFP settings, respectively, when only CFP is present.B: Living Cos-7 cells were incubated with GI262570-FITC. Fluorescenceintensity in the FITC (Exc. 488 nm/Em. 510–530 nm, PMT1)and FRET (Exc. 488 nm/Em. 565–585 nm, PMT2) settings was measuredon at least 100 cells. The SBT ratio is the ratio between the averageFRET and FITC intensities measured in individual cells.emitted by the donor in the FRET channel (F CFP ) andin the donor channel (CFP CFP ), was not constant andincreased with CFP intensity (Fig. 1A). To understandthe causes of such an increase, we tested several possiblefactors. Photobleaching or photoconversion of thefluorophores was not involved as no modification of therelationship between the CFP SBT ratio and CFPintensity was observed after 60 scans of the specimenor after scanning with full laser power (data notshown). We then tested whether this phenomenon wasdependent on the type of fluorophore used by testingthe SBT of FITC. Living cells were incubated with thePPAR ligand GI262570 coupled to FITC, which accumulatesin cellular membranes and in nuclei. Interestingly,when GI262570-FITC was used as a FRET donorwith Cy3, the donor SBT ratio also varied with FITCintensity (Fig. 1B).To determine if the fluctuations of SBTs were due toour instrumentation (Leica TCS SP2 AOBS), we performedthe same experiment with different microscopes.When PPAR-CFP SBT was analyzed on a ZeissLSM510 Meta confocal microscope, using the Metaarray of PMTs, a very similar increase was observedwhen the SBT ratio was plotted as a function of fluorophoreintensity. However, this donor SBT ratio wasconstant on a wide-field microscope using a CCD camera(data not shown). It therefore appeared that thedependency of the SBT ratio on fluorophore intensitywas restricted to confocal microscopes. To further identifythe possible causes leading to this variation, wetested the impact of laser power and PMT gain on SBTratio variations. Variations of CFP SBT ratios were stillobserved when the laser was tuned from 15% to 75% ofits maximum power (data not shown). We then analyzedSBT ratios with four different settings where thegains of the PMTs used for the detection in the donorand FRET channels were set to different voltages.Laser power was adjusted to allow the analysis of thesame batch of cells with each setting (Fig. 2A). Interestingly,although the CFP SBT ratio was independentFig. 2. Influence of PMT gain on the relationship between ECFPSBT ratio and ECFP intensity. Cos-7 cells were transfected with anexpression vector for PPARa-ECFP. Fluorescence intensity in theECFP and FRET settings was measured on at least 100 cells per condition.The percentage of SBT is the ratio between the average FRETand ECFP intensities measured in individual cells. A: The gain andlaser power were set as indicated using PMT1 and PMT2 for ECFP(465–485 nm) and FRET (525–545 nm) detection, respectively. B: Thegain and laser power were set as indicated using PMT2 both for ECFPand FRET detection.of CFP intensity at low PMT gains (500 V donor/530VFRET), this ratio increased with CFP intensity athigher gains. Similar results were obtained when onlythe donor or the FRET gain were changed, or whenboth gains were changed and laser power was kept constant(data not shown).These results suggested that problems in PMT linearitycould account for the observed dependency ofSBT ratios on fluorophore intensity. While it is moreconvenient to use two different PMTs for the detectionin the donor and FRET channels, we investigatedwhether the phenomenon could also be observed withone PMT only. When the same PMT was used for boththe donor and FRET channels, the SBT ratio was con-
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PixFRET, an ImageJ plug-in for FRET calculation ... - ResearchGate

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