Guide for doing FRAP with Leica TCS SP2 - Biocenter

Aug.2004No. 18

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

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

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

FRAPEstimation ofparametersCalculation of the mobile fractionIn an “artificial” in vitro experiment, such as the diffusionof FD 464 in aqueous solution all particles will besubject to Brownian motion and therefore spread bydiffusion. However, in real cells the situation can bemore complicated, because biomolecules/proteinsinteract (bind to) each other. This can lead to a substantialfraction of the labelled molecule, which isbound and therefore does not diffuse, the so-calledimmobile fraction (1 – M f , see below). Only the mobilefraction M f can move. Figure 4 illustrates the effect ofan immobile fraction on the FRAP curve.Calculation of t 1/2The time it takes for the fluorescence to recover to50% of the asymptote (plateau) intensity is called recoveryhalf-time, t 1/2 . It can be used to compare relativerecovery rates, if it is not possible to estimate Dby fitting a diffusion equation to the data. t 1/2 can beeither determined visually from a graph plotting fractionalfluorescence vs. time or by solving (comp.Snapp et al. 2003):F(t) = 100 x (F 0 + F inf (t/t 1/2 )) / (1+(t/t 1/2 )) (3)Note: The recovery half-time strongly depends on thebleach geometry as well as numerous other experimentalparameters (see Table “Experimental Conditions”).In order to compare experiments with eachother they should be conducted with identical conditions,bleach ROI geometry and ROI size.Estimation of the diffusion coefficient DInstead of using equation (3) one could fit an exponentialfunction as empirical approach to the dataset:Figure 4Only the mobile fraction can diffuse. As a resultthe fluorescence level becomes lowerthan 1 (100%).The mobile fraction M f can be calculated directlyfrom the integrated intensities within the bleach ROI(ROI 1 in Figure 5, raw) and a ROI including the wholecell (ROI 2 in Figure 5, raw) according to:With F precell = prebleach intensity within the whole cellF infcell = postbleach intensity (whole cell) at equilibriumF pre = bleach ROI prebleach intensityF(0) = bleach ROI intensity at time 0Note: The first term in eqn. (2) contains a correctionfor photobleaching during the acquisition of the postbleachimages. The latter leads to a fluorescenceloss over time, so the equilibrium intensity would beunderestimated. Without this correction the recoverycould not reach 100 % even with M f = 100%. If a“correction for fluorescence loss due to photobleaching”was performed already (see above), the firstterm eqn. (2) has to be omitted.(2)y = a ∗ (1-exp(-t/τ)) + c (4)This is implemented in the Leica FRAP wizard andyields the time constant t of the recovery as well asthe recovery half-time t 1/2 :t 1/2 = τ ∗ ln 2 (5)In the original publication by Axelrod et al. (1976) asomewhat complicated expression is given for thetemporal behaviour of fluorescence recovery. In thecase the nature of the transport is pure diffusion theysuggest to use the following simplified equation toestimate the diffusion coefficient:D = 0.88 ∗ w 2 /(4 t 1/2 ) (6)With w ... radius of bleached areaHowever, this equation makes the assumptions thatthe bleached area is a spot (disc) and that diffusionoccurs only laterally (in 2D as in membranes). In someexperiments these assumptions may not apply. Insuch a case calculations will only serve as a roughestimation.8Confocal Application Letter

FRAPNote: For fitting eqn 4 to the data it must be normalizedand plotted with the time point of the first postbleachimage t 0 = 0 s.ModelingMore accurate estimates of D can be achieved by fittinga mathematical model numerically to the imageseries recorded (In this case not only the integratedROI intensities, but the whole image data are used).One such simulation for recovery of a strip bleach ROIhas been published by Siggia et al. (2000). Other simulationpackages dealing with diffusion or reactive flowscan be suitable, too, but usually require a considerableamount of work to implement a simulation tailored forlive cell image series. The authors plan to publish soona web-based tool for quantitative FRAP analysis ontheir homepage (http://www.dkfz.de/ibios/index.jsp).T..absolute temperatureη..Viscosity of solventr Η ..hydrodynamic radius of solute moleculeswhere r Η can be estimated using the empirical equation(Braeckmans et al. 2003):r Η = 0.015 ∗ Μ w0.53 (8)WithΜ w ..molecular weightNote: This is only the case for dextransFrom the D obtained the characteristic relaxation timeτ and the recovery half-time t 1/2 were estimated usingD = w 2 /(4τ). Since two approximations have been madeto obtain these parameters, they will be rather inaccurateand are therefore spelled in parentheses.Evaluation of demoexperimentsIn this section our showcase examples, namely FD464 and the H1-GFP cell, will be examplarily evaluated.FD 464Figure 5, upper row, contains integrated fluorescenceintensities for the bleach ROI (green) and a ROI comprisingthe whole cell (purple). The “whole cell” ROI(purple) covers the complete field of view. In the caseof FD464 it was not possible to define a ROI for backgroundintensity, like in H1-GFP (cyan). For backgroundsubtraction an image was taken using therespective photomultiplier with lasers turned to 0%.Table 1 summarizes the results.In the left column the experimental results are given.FD464 theor are the expected values for M f and D. Sinceall FD464 molecules are free to diffuse we expect mobilefraction to be 100%. The experiment shows thatnicely. The 3 % deviation could be attributed to noiseor, more generally, errors during estimation of F infcell .The theoretical D value was estimated using theStokes-Einstein equation,D = k ∗ Τ/(6πηr Η ) (7)With k..Boltzmann constantH1-GFPThe green curves in the right column graphs of Figure 5show clearly a much slower recovery rate for the H1-GFP construct under in vivo conditions. Such a slowrecovery can be indicative of molecular interactions(between histone H1 and chromatin in this case).ROI2 in H1-GFP stays relatively constant, but the intensityincreases by a few percent towards the end ofthe experiment. In general, such an increase couldindicate specimen movement or flux from image planesabove or below the one observed. Such effects cansometimes complicate the quantitative analysis of aFRAP experiment. The corrected curves in the middlerow of Figure 5 demonstrate the correction for fluorescenceloss during acquisition (needed for calculationof M f ) using the whole cell ROI. The bottom row containsthe normalized bleach ROI data together with asingle exponential fit, similar to the fit used by LCS tocalculate the time constant of the recovery. Table 1summarizes the results. Note, that the time constantdepends (among other parameters) on the bleach ROIgeometry, as shown by comparing an arbitraryly shapedROI in Figure 5 and a circular ROI in Figure 6. The timeconstants determined here lie within about 10% deviationof previously published results for H1-GFP (Misteliet al. 2000). If more than a rough estimate is desired,an appropriate model should be chosen (comp. Braeckmanset al. 2003). The same holds true for the timeconstants (D eff ) estimated for the FITC-dextran solution.Confocal Application Letter 9

corrected rawFRAPFD464H1-GFPFigure 5: to be continued on page 1110Confocal Application Letter

normalizedFRAPFigure 5Recovery curves with intensity plotted versus time [s]. FITC-dextran (FD464) and live cells (H1-GFP). Upper row with time integrated ROI intensities (raw) and prebleach (Pre), immediate postbleach(Post1) and equilibrium images (Post3). Bleach ROI shown in green, whole cell: ROI 2, unbleached part of the cell in orange and background intensity in cyan. Middle row compares datacorrected for fluorescence loss (red) with raw data. Bottom row shows normalized data with single exponential fit.Figure 6(A) Recovery curve for bleaching of heterochromatin-rich region ofH1::GFP transfected 3T3 nucleus. (B) First postbleach image withROI (circle).FD464 FD464 theor H1-GFP arbitrary ROI H1-GFP circular ROI H1-GFPliteratureM f [%] 103 100 91 – ~ 90t 1/2 [s] 2.3 (1.0) 138.6 59.9 ~ 55τ [s] 3.3 (1.4) 200.9 41.5 –D eff [µm 2 /s] 1.6 3.7 – 0.01 –Table 1Time constants obtained from exponential fit. FD464 theor calculatedusing an estimation of the hydrodynamic radius and viscosity as determinedin Breackmans et al. (2003). H1-GFP literature value fromMisteli et al. (2000). Results for our running live cell example (H1-GF-P arbitrary ) as well as another cell bleached with a circular ROI (H1-GFP circular ) are given.τ: Time constant of recovery (calculated by LCS, circular ROI)t 1/2 : Half-life of recovery (calculated by LCS, circular ROI)D eff : Effective diffusion coefficient (Axelrod et al. 1976)Confocal Application Letter 11

FRAPFinal RemarksWe would like to recommend beginners, who want topractice FRAP experiments, to use a system similar tothe FITC-dextrans as demonstrated here before tryinglive cells. Keep in mind that in particular the bleacharea size, the laser intensities, FRAP modes, bleachformat, scan speeds as well as the iteration numbersand intervals for bleach and post-bleach acquisitionhave to be optimized for each cell type and protein ofinterest. The results given here were obtained byevaluating just one data set. For meaningful resultsthe experiment should be repeated at least 10-20 timesto get an estimate for the standard deviation. Repeatingthe FRAP experiment using the same cell andbleach ROI can also reveal potential phototoxicity, ifthe results are not reproducible. In such a case theFRAP protocol has to be further optimized (s.a.decreasing the laser power).References1. Phair, R.D., Misteli, T. (2000) High mobility of proteins in the mammaliancell nucleus. Nature. 404:6042. Beaudouin, J., Gerlich, D., Daigle, N., Eils R., Ellenberg, J. (2002)Nuclear Envelope Breakdown Proceeds by Microtubule-InducedTearing of the Lamina Cell 108:83-963. Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., Webb, W.W.(1976) Mobility measurement by analysis of fluorescence photobleachingrecovery kinetics. Biophys. J. 16:1055-10694. Phair, R.D., Misteli, T. (2001) Kinetic modelling approaches to invivo imaging. Nat Rev Mol Cell Biol. 2:898-9075. Snapp, E.L., Altan, N., Lippincott-Schwartz, J. (2003) Measuringprotein mobility by phtobleaching GFP chimeras in living cells.Curr. Prot. Cell. Biol. 21.1-21.1.246. Ellenberg., J., Siggia, E.D., Moreira, J.E., Smith, C.F., Presley, J.F.,Worman, H.J., Lippincott-Schwartz, J. (1997) Nuclear membranedynamics and reassembly in living cells: Targetting of an innernuclear membrane protein in interphase and mitosis. J. Cell. Biol.138:1193-12067. Siggia, E.D., Lippincott-Schwartz, J., Bekiranov, S. (2000) Diffusionin inhomogenous media: Theory and simulations applied to a wholecell photobleach recovery. Biophys. J. 79:1761-17708. Lippincott-Schwartz, J., Altan-Bonnet, N., Patterson, G.H. (2003)Photobleaching and photoactivation:following protein dynamicsin living cells. Nature Cell Biology Suppl:S7-S149. Braeckmans, K., Peeters, L., Sanders, N.N., De Smedt, S.C.,Demeester, J. (2003) Three-Dimensional Fluorescence Recoveryafter Photobleaching with the Confocal Scanning Laser Microscope.Biophysical Journal 85:2240–225210.Misteli, T., Gunjan, A., Hock, R., Bustink, M., Brown, D.T. (2000)Dynamic binding of histone H1 to chromatin in living cells. Nature408:877-880Copyright © Leica Microsystems Heidelberg GmbH • Am Friedensplatz 3 • D-68165 Mannheim, Germany • Tel. +49 (0) 6 21-70 28-0 • E-Mail: clsm.support@leica-microsystems.com LEICA and the Leica Logo are registered trademarks of Leica IR GmbH.Order no. of the edition: 1593104005 • August 2004@ www.confocal-microscopy.com