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FRAP

IntroductionAmong all photobleaching experiments which havebeen described, fluorescence recovery after photo-bleaching (FRAP) is the most popular. It employs irra-diation of a fluorophore in a living sample with a shortlaser pulse to degrade it and thereby abolish fluores-cence followed by time-resolved image recording ofthe sample. If the fluorophore (or a so-called mobilefraction of it, see below) is able to freely diffusethrough the sample a recovery of fluorescence canbe observed. Photobleaching experiments can beconducted with modern laser scanning microscopes,where the laser is used at high intensity for bleachingand low intensity for image recording. The LCS soft-ware provided by Leica contains a FRAP applicationwizard that guides the user through the steps of aFRAP experiment. This wizard will be used in theexamples below.

Qualitative vs.Quantitative FRAPFRAP experiments have been applied to study thedynamics within and between subcellular compart-ments (Phair and Misteli, 2000) as well as changes intheir morphology (Beaudouin et al. 2002).

In this way qualitative information about intracellulartransport processes can be rendered in live cells. If atime series is recorded immediately after the bleach-ing pulse at high rate (> 5 s-1) a quantitative evaluationbecomes possible. Parameters that can be obtainedare the diffusion coefficient D, and the mobile fract-ion Mf. Alternatively, instead of D the characteristichalf-time of the recovery t1/2, can be retrieved from

the data. In fact, t1/2 is reciprocally proportional to Dwith the assumptions made by Axelrod et al. (1976).See also “data processing” in this paper. For review,see Phair and Misteli (2001), Snapp et al. (2003) or Lip-pincott-Schwartz et al. (2003).

PreparationThe most common way to fluorescently tag proteinsin living cells is the GFP technology (green fluores-cent protein cloned from the jelly fish A. victoria). Ofthe different spectral GFP mutants EGFP is the best,because of its high quantum yield, its low tendency ofphotobleaching as well as its relative photostabilityduring post-bleach image acquisition. Cells both stablyor transiently expressing an EGFP-tagged version ofyour protein of interest (ypi) are suitable for FRAPexperiments. Transiently transfected cells are oftenused 16 – 48 hours post transfection. Here the optimaltime for the experiment has to be determined empiri-cally depending on the construct and the cell type orculture conditions. To maintain appropriate cultureconditions for most cells it is necessary to have atleast a heatable stage, ideally contained within amoisturized and CO2-controlled box. Prerequisite forFRAP experiments is a confocal laser scanning micros-cope capable of modulating the beam intensity duringthe scan. The Leica TCS SP2 capability of switchingbetween two laser intensities enables even moresophisticated experiments.

Establish experimental conditions1. Prepare the cells in an imaging chamber 16 – 48

hours prior to the FRAP experiment. 2. Set up microscope and prewarm the stage. Let the

UV-lamp (for optical control) and the lasers warmup until steady.

Fluorescence recovery after photobleachingwith the Leica TCS SP2

Authors: Constantin Kappel and Roland Eils, German Cancer Research Center (DKFZ), Div. Theoretical Bioinformatics,Im Neuenheimer Feld 580, 69120 Heidelberg, http://www.dkfz.de/ibios/index.jsp

Confocal Application Letter2


FRAP

3. 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 bidi-rectional scanning if appropriate. For freely diffus-ing (i.e. non-binding) molecules the fastest scan speed 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. Further-more, 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. Double-

check or 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 read-out of recovery is done between lines. This means,your recovery readout is closest possible to thereal zero time (t0) of the postbleach intensity.The FlyMode combines both, the bleach scan andfirst image scan after bleaching. Bleaching is perfor-med during fly forward using ROI Scan features and high laser power. During fly back, the laser intensity

is set to imaging values (AOTF switching withinmicro-seconds). Thus, the first image is acquiredsimultaneously with the bleaching frame. And con-sequently, 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 normaliza-tion of 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 im-portant information about the fluorescence reco-very is contained within the data immediately afterthe bleach pulse. Therefore the first post-bleachstep (Fig. 1) should be recorded at the highest pos-sible rate. 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 bidi-rectional scanning. 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


FRAP

6. Press next. Now the laser intensity for bleachinghas to be set. EGFP or FITC have an absorbance maximum near the 488 nm line of an Ar-Ion laser.Additionally activating the 458 and 476 nm laserlines can increase the bleaching depth. The bleach-ing depth is the ratio between the post-bleach in-tensity F0 and the prebleach intensity within thebleached region. F0 should be close or equal to background intensity, otherwise the curvature of the 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 estab-lishing the FRAP procedure using a fixed sample canhelp (Snapp et al. 2003).

7. Define a region of interest (ROI) for bleaching. It is always a good idea to save the ROI for quantifica-tion later 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.

Note: There are different geometries for bleach ROIsavailable in LCS. The most widely used ones are spotphotobleaching (circular ROI) and strip photoblea-ching (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 photo-bleaching was 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, characteri-stic half time and rate of the recovery. However thisfit result has to be considered preliminary, becauseat this point no background subtraction, bleaching correction 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 pro-cessing or statistics software (i.e. Excel, Mathema-tica, Matlab or others).

Note: In order to be able to repeat the experimentwith exactly the same settings (i.e. for repeated photo-bleaching of the same cells as a test for phototoxicityand reproducibility) it is recommended to save thesettings.

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 pro-cedure we will use a 464 kD dextran labelled withfluorescein-isothiocyanate (FITC-dextran, FD464), whichis commercially available from most laboratory sup-pliers. 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.


Figure 1Live cell: Fluorescence reco-very plotted versus time in 8-bitformat (i.e. max = 255). Timeintegrated grey values (inten-sities) of bleach ROI (green),whole cell ROI (purple), unble-ached part of cell (orange,more information, see text).Three iterations with bleachintensity were applied (bet-ween arrows). The backgro-und intensity was not takeninto consideration in this ex-ample . Note: Only in the Fly-Mode you are able to monitorthe fluorescence decay duringbleaching.


FRAP

Photobleaching 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, Sond-heim, Germany), covered with a coplanar coverslipand sealed with acetone-free nail polish.

Experimental conditions were:Ojective 63x NA 1.4 Oil Scan mode xytScan speed 1400 HzExcitation wavelength 488 nmEmission range 498-600 nmFormat 256 x 256Zoom 8 Laser power/potentiometer (Ar/KrAr) ~75 %AOTF (imaging) 488 nm 1.5 %AOTF (bleaching) 488 nm 100%ROI geometry CircleROI diameter 9.2 µmPrebleach 20 x 294 msBleach 3 x 294 msPost 1 15 x 294 msPost 2 60 x 1 sPost 3 50 x 5 s

After having established experimental conditions wenow have to optimize the bleach pulse as well as thenumber and rate of post-bleach images. For this pur-pose the 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 iterati-ons will be used. This way we can optimize the lengthof the bleach pulse and can now monitor the early on-set of 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.

Note: 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 ana-lysis). 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 re-sulting in 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 subse-quent images can be minimized by switching on“complete burst” mode. With this setting image ac-quisition is at maximum speed. Therefore the screenwill not show any live images during recording.

FRAP of a nuclear protein in live cellsHistone H1 – tagged with EGFP (contruct kindly provi-ded by T. Misteli) was transfected into 3T3 NIH mousefibroblast cells. A FRAP experiment was conductedwith the following conditions:

Experimental conditions were: Ojective 63x NA 1.4 Oil Scan mode xytScan speed 1400 HzExcitation wavelength 488 nmEmission range 498-600 nmFormat 256x256Zoom ~ 8 Laser 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 geometry PolygonROI size (enclosing rectangle) 15.3 µm x 4.1 µmPrebleach 20 x 294 msBleach 3 x 294 msPost 1 40 x 294 msPost 2 30 x 2 sPost 3 24 x 10 s



FRAP

Figure 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 t1/2 and therecovery rate are only rough estimates, because thedata has not been corrected. There are three neces-sary corrections: 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 ac-tual data (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 misloca-tion of 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 stai-ned in 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.

Correct for fluorescence lossdue to photobleachingWe have to distinguish two kinds of photobleaching:Firstly, the intended photobleach with full laser inten-sity during the bleach pulse, secondly, photobleach-ing during post-bleach acquisition. While the formerphotobleaching is intended, the latter is inherent tothe imaging process and can lead to some considera-ble loss of total fluorescence. Therefore, the fluores-cence intensity can never recover to 100%, even if allfluorophore molecules were mobile (see “Calculationof mobile fraction”). It is possible to correct for this amount of photobleaching by multiplying every element

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).



FRAP

of the data set by (Fprecell/Finfcell), where Fprecell meansprebleach intensity within the whole cell, Finfcellstands 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% fluo-rescence loss.

Note: Alternatively, a second cell, which does nottake part in the FRAP experiment, can be used as anindependent control. In this case the ratio Fprecell/Finfcell would be determined for this cell.

Note: Fprecell/Finfcell can only be accurately determi-ned for a closed compartment without exchangewith the surroundings within the time-scale of theexperiment (s.a. the nucleus in our live cell exam-ple). In spite of this limitation the data from the FITC-dextran experiment was used here, for illustrationpurposes only.

NormalizationNormalization means to rescale the images in a waythat represents plateau fluorescence as 100% (frac-tional fluoresence intensity) in order to make severalimage series mutually comparable as well as to ena-ble us to readily estimate the relaxation half-time t1/2.

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 norma-lized to 100%. This type of normalization is suitable tographically determine the mobile fraction from nor-malized data, while the relaxation half-time can not.This method can also be applied to incomplete reco-veries (i.e. fluorescence intensity has not reached aplateau value.).

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 equilibrium

Using 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.

Figure 3Schematic cell expressing GFP mainly in the nucleus. A region is ble-ached in 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).



FRAP

Calculation of the mobile fractionIn an “artificial” in vitro experiment, such as the diffu-sion of 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 sub-stantial fraction of the labelled molecule, which isbound and therefore does not diffuse, the so-calledimmobile fraction (1 – Mf, see below). Only the mobilefraction Mf can move. Figure 4 illustrates the effect ofan immobile fraction on the FRAP curve.

The mobile fraction Mf 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:

Note: The first term in eqn. (2) contains a correctionfor photobleaching during the acquisition of the post-bleach images. The latter leads to a fluorescenceloss over time, so the equilibrium intensity would beunderestimated. Without this correction the reco-very could not reach 100 % even with Mf = 100%. If a“correction for fluorescence loss due to photoblea-ching” was performed already (see above), the firstterm eqn. (2) has to be omitted.

Calculation of t1/2The time it takes for the fluorescence to recover to50% of the asymptote (plateau) intensity is called re-covery half-time, t1/2. It can be used to compare rela-tive recovery rates, if it is not possible to estimate Dby fitting a diffusion equation to the data. t1/2 can beeither determined visually from a graph plotting frac-tional fluorescence vs. time or by solving (comp.Snapp et al. 2003):

F(t) = 100 x (F0 + Finf(t/t1/2)) / (1+(t/t1/2)) (3)

Note: The recovery half-time strongly depends on thebleach geometry as well as numerous other experi-mental parameters (see Table “Experimental Condi-tions”). In order to compare experiments with eachother they should be conducted with identical condi-tions, bleach ROI geometry and ROI size.

Estimation of the diffusion coefficient DInstead of using equation (3) one could fit an expo-nential function as empirical approach to the dataset:

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 t1/2:

t1/2 = 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∗w2/(4 t1/2) (6)With w ... radius of bleached area

However, 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.

Figure 4Only the mobile fraction can diffuse. As a re-sult the fluorescence level becomes lowerthan 1 (100%).


Estimation ofparameters

(2)

With Fprecell = prebleach intensity within the whole cellFinfcell = postbleach intensity (whole cell) at equilibriumFpre = bleach ROI prebleach intensityF(0) = bleach ROI intensity at time 0


FRAP

Note: For fitting eqn 4 to the data it must be normalizedand plotted with the time point of the first postbleachimage t0 = 0 s.

ModelingMore accurate estimates of D can be achieved by fit-ting a 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 simu-lation packages 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).

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 com-prising the 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 back-ground intensity, like in H1-GFP (cyan). For back-ground subtraction 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.FD464theor are the expected values for Mf and D. Sinceall FD464 molecules are free to diffuse we expect mo-bile fraction to be 100%. The experiment shows thatnicely. The 3 % deviation could be attributed to noiseor, more generally, errors during estimation of Finfcell.The theoretical D value was estimated using theStokes-Einstein equation,

D = k∗Τ/(6πηrΗ) (7)With k..Boltzmann constant

T..absolute temperatureη..Viscosity of solventrΗ..hydrodynamic radius of solute molecules

where rΗ can be estimated using the empirical equa-tion (Braeckmans et al. 2003):

rΗ = 0.015∗Μw0.53 (8)

With Μw..molecular weight

Note: This is only the case for dextrans

From the D obtained the characteristic relaxation timeτ and the recovery half-time t1/2 were estimated usingD = w2/(4τ). Since two approximations have been madeto obtain these parameters, they will be rather inac-curate and are therefore spelled in parentheses.

H1-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 in-tensity increases by a few percent towards the end ofthe experiment. In general, such an increase couldindicate specimen movement or flux from image pla-nes above 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 fluore-scence loss during acquisition (needed for calculationof Mf) using the whole cell ROI. The bottom row con-tains the 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% devia-tion of previously published results for H1-GFP (Misteliet al. 2000). If more than a rough estimate is desired,an appropriate model should be chosen (comp. Braeck-mans et al. 2003). The same holds true for the timeconstants (Deff) estimated for the FITC-dextran solution.



FRAP


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FD464 H1-GFP

Figure 5: to be continued on page 11


FRAP

Table 1Time constants obtained from exponential fit. FD464theor calculatedusing an estimation of the hydrodynamic radius and viscosity as de-termined in Breackmans et al. (2003). H1-GFP literature value fromMisteli et al. (2000). Results for our running live cell example (H1-GF-Parbitrary ) as well as another cell bleached with a circular ROI (H1-GFPcircular) are given.

FD464 FD464theor H1-GFParbitrary ROI H1-GFPcircular ROI H1-GFPliterature

Mf [%] 103 100 91 – ~ 90t1/2 [s] 2.3 (1.0) 138.6 59.9 ~ 55τ [s] 3.3 (1.4) 200.9 41.5 –Deff [µm2/s] 1.6 3.7 – 0.01 –

ττ: Time constant of recovery (calculated by LCS, circular ROI)

t1/2: Half-life of recovery (calculated by LCS, circular ROI)Deff: Effective diffusion coefficient (Axelrod et al. 1976)

Figure 6(A) Recovery curve for bleaching of heterochromatin-rich region ofH1::GFP transfected 3T3 nucleus. (B) First postbleach image withROI (circle).


norm

alize

d

Figure 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 post-bleach (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.


FRAP

We 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 by evaluating just one data set. For meaningful resultsthe experiment should be repeated at least 10-20 timesto get an estimate for the standard deviation. Repeat-ing the 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 mam-

malian cell nucleus. Nature. 404:6042. Beaudouin, J., Gerlich, D., Daigle, N., Eils R., Ellenberg, J. (2002)

Nuclear Envelope Breakdown Proceeds by Microtubule-Induced Tearing of the Lamina Cell 108:83-96

3. Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., Webb, W.W. (1976) Mobility measurement by analysis of fluorescence photo-bleaching recovery kinetics. Biophys. J. 16:1055-1069

4. Phair, R.D., Misteli, T. (2001) Kinetic modelling approaches to invivo imaging. Nat Rev Mol Cell Biol. 2:898-907

5. Snapp, E.L., Altan, N., Lippincott-Schwartz, J. (2003) Measuringprotein mobility by phtobleaching GFP chimeras in living cells.Curr. Prot. Cell. Biol. 21.1-21.1.24

6. 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. 138:1193-1206

7. Siggia, E.D., Lippincott-Schwartz, J., Bekiranov, S. (2000) Diffusionin inhomogenous media: Theory and simulations applied to a wholecell photobleach recovery. Biophys. J. 79:1761-1770

8. Lippincott-Schwartz, J., Altan-Bonnet, N., Patterson, G.H. (2003)Photobleaching and photoactivation:following protein dynamicsin living cells. Nature Cell Biology Suppl:S7-S14

9. Braeckmans, K., Peeters, L., Sanders, N.N., De Smedt, S.C.,Demeester, J. (2003) Three-Dimensional Fluorescence Recovery after Photobleaching with the Confocal Scanning Laser Micros-cope. Biophysical Journal 85:2240–2252

10.Misteli, T., Gunjan, A., Hock, R., Bustink, M., Brown, D.T. (2000)Dynamic binding of histone H1 to chromatin in living cells. Nature 408:877-880

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@www.confocal-microscopy.com


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