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Kevin,
Could you or another FRAPper elaborate on one of your points?  Re: the
"empirical" methods that you don't recommend, you explain the variability in
photobleaching rates by what seems a reasonable assumption: variability in
viscosity, causing variability in diffusion rates.  In other words, it seems
that such a measurement of diffusion rate is therefore reporting what it is
supposed to report, and the variability can be summarized as unavoidable
variability in cell morphology.  Let me know what I'm missing here.

Even if the variability in photobleaching is not caused by variability in
diffusion rates, what else besides a changing diffusion rate could explain a
change in recovery half-life?  Even if there is more variability in the
empirical method (for whatever reason) than with methods offering
near-instantaneous photobleaching, you should still be able to overcome the
experiment-to-experiment variability by ratioing to the same control in each
experiment, and increasing the # samples.

People here have gotten consistent results when FRAPping with the empirical
method, and of course I would like to know if we misinterpret significant
differences as indicating varying diffusion rates.

CHeers,
Jeff M. Reece
Biomedical Engineer
Confocal Microscopy Center
National Institute of Environmental Health Sciences (NIEHS)
111 Alexander Drive, Bldg, 101, Rm. F219
P.O. Box 12233, MD F2-02
Research Triangle Park, NC  27709
(919) 541-0311
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> -----Original Message-----
> From: Kevin Braeckmans [mailto:[log in to unmask]]
> Sent: Wednesday, November 24, 2004 4:09 AM
> To: [log in to unmask]
> Subject: Re: FRAP experiments
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Dear Jammi,
>
> Calculating a diffusion coefficient from an FRAP experiment
> is usually = based on the fitting of a particular FRAP model
> to the experimental = fluorescence recovery curve. Therefore,
> to obtain a valid diffusion coefficient, it = is of utmost
> importance that your experiment is carried out according to
> the theory of the FRAP model you want to use.
>
> Apart from a few exceptions, most FRAP models indeed assume
> an = instantaneous photobleaching phase, which in fact is the
> assumption of having no = recovery while photobleaching.
> Hence, the the time for photobleaching should be = very short
> (< 5%) compared to your experiment's characteristic recovery
> time. = In actual practice this means that in most cases you
> can only use one = bleaching iteration (although it depends
> on many parameters: scanning speed, = bleach geometry,
> diffusion speed). For longer photobleaching times you will
> get = the 'corona' effect you mentioned, leading in fact to a
> decreased (not
> increased) diffusion coefficient.
>
> As long as you respect this simple but important rule, the
> actual = percentage of photobleaching doesn't matter, at
> least if you are using a good FRAP model. With 'good' I mean
> a FRAP model which takes the amount of photobleaching
> explicitly into account. For such a FRAP model you can
> include this 'photobleaching parameter' as a free fitting
> variable to = obtain its value for each experiment from a
> fitting to the data.
>
> For completeness sake I should mention that there are FRAP
> models which = do not require an instantaneous photobleaching
> phase (see list below). One = is based on Fourier transforms,
> the other on a statistical analysis. The Fourier method is
> very neat in theory, but doesn't seem to work very = well in
> actual practice based on our own experience because it is
> very noise sensitive. Does anyone of the Confocal List have a
> different experience = with this?
>
> Finally, there are also empirical FRAP methods which are
> based on the = idea that if one does exactly the same in each
> sample (same bleach intensity, bleach time and bleach
> geometry) one can get a relative diffusion coefficient by
> comparing the recovery-half-times. While this sounds a
> reasonable assumption in theory, I wouldn't recommend it
> because in = actual practice you will not get the same
> photobleaching result in different samples, as you have
> observed for yourself in your own experiments. The = main
> reason for this is that the photobleaching process is based
> on = photochemical reactions in which many different
> molecules play a role. So for example, = in a high viscosity
> sample you will get less photobleaching compared to a similar
> low viscosity sample because in the latter the molecules are
> diffusing more rapidly, hence increasing the number of
> photobleaching reactions per second, hence leading to more
> photobleaching. And things become even more complicated when
> doing measurements in different = regions of a cell where the
> chemical environment can differ as well.
>
> So as you can see it all comes down to which FRAP model you
> will be = using for calculating the diffusion coefficient. In
> case you have not decided = yet which model to use, below is
> a (non exhautive) list of possible FRAP models/methods.
>
> Hopefully this is of help to you.
>
> Best regards,
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry and Physical Pharmacy
> Ghent University
> Belgium
>
>
> Here's the list.
>
> 1. Axelrod, D., D.E. Koppel, J. Schlessinger, J. Elson, and
> W.W. Webb. = 1976. Mobility measurement by analysis of
> fluorescence photobleaching recovery kinetics. Biophys. J.
> 16:1055-1069.
>
> This fundamental article covers 2D diffusion and 1D flow for
> Gaussian = and uniform spot photobleaching. A more practical
> expression for uniform = spot photobleaching has been derived by:
>
> 2. Soumpasis, D.M. 1983. Theoretical analysis of fluorescence
> = photobleaching recovery experiments. Biophys. J. 41:95-97.
>
> FRAP in 2D with the uniform spot method has been examined for
> 2 = diffusing components as well:
>
> 3. Gordon, G.W., B. Chazotte, X.F. Wang, and B. Herman. 1995.
> Analysis = of simulated and experimental fluorescence
> recovery after photobleaching. = Data for two diffusing
> components. Biophys. J. 68:766-778.
>
> A paper discribing FRAP for a Gaussian spot in case of second
> order photobleaching kinetics:
>
> 4. Bjarneson, D. W., and N. O. Petersen. 1991. Effects of
> second order photobleaching on recovered diffusion parameters
> from fluorescence photobleaching recovery. Biophys. J. 60:1128-1131.
>
> A 3D extension of the Gaussian spot has been proposed by:
>
> 5. Blonk, J.C.G., A. Don, H. Van Aalst, and J.J. Birmingham.
> 1993. Fluorescence photobleaching recovery in the confocal
> scanning light microscope. J. Microsc. 169:363-374.
>
> And a similar 3D extension of the uniform spot was recently
> derived by = us. The model is also applicable to bleaching
> disk-shaped geometries on = confocal scanning microscopes:
>
> 6. Braeckmans, K., L. Peeters, N. N. Sanders, S. C. De Smedt,
> and J. Demeester. 2003. Three-dimensional fluorescence
> recovery after photobleaching with the confocal microscope.
> Biophys. J. 85:2240-2252.
>
> A numerical approach for the 2D uniform spot (or disk) has
> been = presented
> by:
>
> 7. Lopez, A., L. Dupou, A. Altibelli, J. Trotard, and J.
> Tocanne. 1988. Fluorescence recovery after photobleaching
> (FRAP) experiments under conditions of uniform disk
> illumination. Biophys. J. 53:963-970.
>
> A numerical approach for 2D FRAP in a diffraction limited
> line segment = was developed by:
>
> 8. Wedekind, P., U. Kubitscheck, O. Heinrich, and R. Peters.
> 1996. Line-Scanning microphotolysis for diffraction-limited
> measurements of lateral diffusion. Biophys. J. 71:1621-1632.
>
> and later in 3D as well:
>
> 9. Kubitscheck, U., P. Wedekind, and R. Peters. 1998.
> Three-dimensional diffusion measurements by scanning
> microphotolysis. J. Microsc. = 192:126-138.
>
> A statistical evaluation of 2D FRAP for arbitrary radially
> symmetric bleaching geomteries has been presented as well.
> This method has the advantage of being independent of the
> bleaching kinetics and possible recovery during bleaching. A
> disadvantage is that it does not allow to calculate the
> immobile fraction independently.
>
> 10. Kubitscheck, U., P. Wedekind, and R. Peters. 1994.
> Lateral diffusion measurements at high spatial resolution by
> scanning microphotolysis in a confocal microscope. Biophys.
> J. 67:948-956.
>
> A method for 2D FRAP having essentially the same advantages
> and disadvantages, relies on a calculation involving Fourier
> transforms of = the recovery images. An additional advantage
> is that the bleaching geometry = is completely arbitrary. The
> following two articles explain how the = technique
> works:
>
> 11. Tsay, T., and K.A. Jacobson. 1991. Spatial Fourier
> analysis of video photobleaching measurements, principles and
> optimization. Biophys. J. 60:360-368.
>
> which should be read in combination with
>
> 12. Berk, D.A., F. Yuan, M. Leunig, and R. K. Jain. 1993.
> Fluorescence photobleaching with spatial Fourier analysis:
> measurement of diffusion = in light-scattering media.
> Biophys. J. 65:2428-2436.
>
> A method for the analysis of a distribution of diffusion
> coefficients = (in stead of just 1 or 2) has been described by:
>
> 13. Periasamy, N., and A. S. Verkman. 1998. Analysis of
> fluorophore diffusion by continuous distributions of
> diffusion coefficients: = Application to photobleaching
> measurements of multicomponent and anomalous = diffusion.
> Biophys. J. 75:557-567.
>
> A method for measuring relative diffusion coefficients by FRAP:
>
> 14. Kao, H. P., J. R. Abney, and A. S. Verkman. 1993.
> Determinants of = the translational mobility of a small
> solute in cell cytoplasm. J. Cell = Biol. 120:175-184.
>
> and later articles by the same authors. For a review of their
> methods =
> see:
> Verkman, A. S. 2003. Diffusion in cells measured by
> flourescence = recovery after photobleaching. In
> Biophotonics, Part A: Methods in Enzymology, = Volume 360. G.
> Marriott and I. Parker, editors. Academic Press, New York, pp.635-648.
>
> 1D FRAP in a limited volume based on Fourier transforms was
> derived by:
>
> 15. Elowitz,M.B.; Surette,M.G.; Wolf,P.E.; Stock,J.B.;
> Leibler,S. 1999. Protein mobility in the cytoplasm of
> Escherichia coli. J. Bacteriology. 181:197-203.
>
> A FRAP model for 1D diffusion including association and dissociation:
>
> 16. Carrero, G., D. McDonald , E. Crawford, G. de Vries, and
> M. J. = Hendzel. 2003. Using FRAP and mathematical modeling
> to determine the in vivo = kinetics of nuclear proteins.
> Methods 29:14-28.
>
> A paper describing a 'Fringe Pattern Photobleaching and
> Recovery' = method, as well as a FRAP method relying on a
> uniformly bleached long line segment:
>
> 17. Cheng, Y., R. K. Prud'homme, and J. L. Thomas. 2002.
> Diffusion of mesoscopic probes in aqueous polymer solutions
> measured by fluorescence recovery after photobleaching.
> Macromolecules 35:8111-8121.
>
> An other paper describing FRAP in case of a long line segment:
>
> 18. Dietrich, C., R. Merkel, and R. Tampe. 1997. Diffusion
> measurement = of fluorescence-labeled amphiphilic molecules
> with a standard fluorescence microscope. Biophys. J. 72:1701-1710.
>
> A recent paper describing a FRAP technique using continuous
> spot photobleaching allowing to calculate dissociation and
> residence times at binding sites in addition to the diffusion
> coefficient:
>
> 19. Wachsmuth,M.; Weidemann,T.; Muller,G.;
> Hoffmann-Rohrer,U.W.; = Knoch,T.A.; Waldeck,W.; Langowski,J.
> 2003. Analyzing intracellular binding and = diffusion with
> continuous fluorescence photobleaching. Biophys. J. 84:3353-3363.
>
>
>
> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List
> [mailto:[log in to unmask]] = Namens Narasimham Jammi
> Verzonden: woensdag 24 november 2004 0:59
> Aan: [log in to unmask]
> Onderwerp: FRAP experiments
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Hi all,
>  I have a question regarding the number of bleach iterations
> one would = want to use in a typical FRAP experiment. I
> understand that one would want to minimize the number of
> bleach iterations (preferrably limit it to 1 = bleach at 100%
> laser intensity) or else fall prey to the 'corona' effect at
> the ROI, thereby leading to an increase (flawed) in the
> diffusion = coffecients. However, bleaching just once leads
> to uneven decrease in fluorescence intensities over different
> samples (for eg, the intensity on ROI goes = from 100% to 60%
> in sample 1, sample 2: 100% to70%, sample 3: 100% to 80% =
> etc). Does this make a difference in interpreting the data?
>
> thanks
> -Jammi
>