Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Jim,

> But surely, if the area of the bleach has been seriously disrupted,
> the 'diffusion rate' measured will not tell you much about the Diff 
> Rate in the absence of the disruption
> 
> How do you check for this?

You do have a point there. Without wanting to avoid your question, we are
only just starting our first intracellular FRAP measurements, so I don't
have any practical experience with that. Untill now we have been using FRAP
in a pharmaceutical context on extracellular matrices and systems for
controlled drug release.

However, as far as I can see, I don't expect it to be a problem. In FRAP one
uses fast photobleaching fluorophores (as opposed to imaging confocal
microscopy), so in fact we do not use an excessivly high laser power for
photobleaching. At the contrary, a basic rule is to use as little laser
power as possible to avoid fluorophore saturation as much as possible.

Some quantitative numbers to give you an idea. We do quantitative FRAP with
a low NA lens (NA 0.3), so the radius of the diffraction limited spot is
around 1 micron and its area is approx. 3 micron^2. For R-phycoerythrin
labeled samples we typically use a laser power of 0.5 to 1 mW at the
objective lens. For fluorescein constructs it is rather 2 mW. The local
irradiance when  photobleaching is therefore between 0.2 and 0.7
mW/micron^2.

For comparison, in confocal microscopy one usually uses more photostable
fluorophores and high NA lenses (NA 1.4 for oil-immersion). The diffraction
limited spot for such a lens has an area of approx. 0.15 micron^2. I have
seen confocalists use 50 microWatt and more (!) on weakly stained cells to
get an image. This actually means an irradiance of 0.3 mW/micron^2 as well,
which is of the same order. And I didn't see them worrying about destroying
the structure of their cells either...

In addition there is ofcoure the photobleaching time which should be
considered to get a fair comparison. However, since nowadays most FRAP
experiments are carried out on confocal microscopes with a scanning laser
beam, the comparison above still holds. The situation might be a little
different for FRAP methods in which photobleaching is still done with a
stationary light beam (although I think this approach is rather getting
obsolete). In that case, the total energy received might actually be
substantially larger compared to confocal microscopy, depending on the
photobeaching time. But then I would like to compare with FCS, where
measurements are made with a laserbeam which remains stationary for
sometimes several minutes! To the best of my knowledge I haven't come across
issues in literature describing destructive effects of FCS measurements on
live samples.

Nevertheless, your criticism is legitimate and care should be taken. I think
the only real answer is that one should probe the interior of the cell in
more than one way. For example, we are also using FCS and are planning on
using single particle tracking as well. I think it is the combination of
such complementary techniques which will give the most complete insight and
best controll of the individual experiments.

Best regards,

Kevin

> -----Oorspronkelijk bericht-----
> Van: James Pawley [mailto:[log in to unmask]]
> Verzonden: vrijdag 26 november 2004 20:18
> Aan: Kevin Braeckmans
> Onderwerp: Re: FRAP experiments
> 
> 
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Dear Jeff,
> >
> >First of all, it is great that you get consistent results using the
> >empirical FRAP method; in the end it is the experiment alone which 
> >gives the final proof. To be clear, I didn't mean to say it doesn't 
> >work, it is just that, according to my understanding, there 
> may be some
> >pitfalls associated with the method which might be hard to avoid or
> >even notice, certainly for someone relatively new to 
> quantitative FRAP
> >experiments.
> >
> >I will try to explain my reservations about the method, which are
> >purely from a theoretical point of view. But I may very well be 
> >mistaking, so please correct me if I am wrong; it is always 
> >advantageous to gain new insights.
> >
> >In a FRAP experiment, the fluorescence recovery happens after
> >photobleaching because of diffusion. In general, the shape of the 
> >recovery curve will depend on 1) the amount of 
> photobleaching, 2) the
> >bleach geometry (shape + size), 3) sample space (e.g. 2D or
> 3D) and 4)
> >the diffusion speed. Therefore, to obtain the diffusion coefficient
> >from the recovery curve, one has to take the amount of 
> photobleaching,
> >the bleach geometry and the sample space carefully into account. And
> >this is exactly what is done by the various FRAP models 
> which are based
> >on a solution of Fick's second law for those particular boundary
> >conditions.
> 
> 
> 
> But surely, if the area of the bleach has been seriously disrupted,
> the 'diffusion rate' measured will not tell you much about the Diff 
> Rate in the absence of the disruption
> 
> How do you check for this?
> 
> Jim P.
> 
> 
> >The empirical method, at the other hand, is based on the assumption
> >that if one keeps all boundary conditions the same for each 
> experiment,
> >a change in the recovery curve can only be due to a change
> in diffusion
> >coefficient. Therefore, by comparing the recovery curves from two
> >experiments one can obtain a relative change in diffusion 
> coefficient.
> >As already said, this sounds like a very reasonable
> approach, and very
> >easy to carry out as well.
> >
> >In actual practice, howver, it seems to me that it will be very
> >difficult (if possible at all) to obtain the same boundary 
> conditions
> >in two different samples. My major concern is the condition of the
> >amount of photobleaching which has to be exactly the same, 
> because for
> >the same laser intensity, the amount of photobleaching can be very
> >different for a low and a high viscosity sample (see my previous 
> >e-mail). In addition, if the chemical environment is 
> different for both
> >samples, the photobleaching rate and hence the amount of
> photobleaching
> >can be very different as well. According to the reasoning
> above, if the
> >amount of photobleaching is different, the two recovery
> curves cannot
> >be compared directly in terms of a change in diffusion coefficient
> >alone.
> >
> >In addition, also the following two issues are critical to obtain
> >correct values for the diffusion coefficient.
> >
> >1. For the same photobleaching time, more recovery will have
> happened
> >for a fast diffusing sample compared to a more slowly
> diffusing one. As
> >a consequence, the bleached geometry in the fast sample will
> generally
> >be expanded compared to the slow sample. Again, if both bleach
> >geometries are not exactly the same, the recovery curves cannot be 
> >compared any more. Therefore it will be of critical 
> importance that the
> >condition of an instantaneous bleach phase is respected.
> (This is the
> >same for most of the 'physical' FRAP models, though.)
> >
> >2. The sample space in which diffusion takes place should be
> the same
> >for both samples. While this is not a problem when doing
> experiments in
> >3D samples such as solutions etc., one can imagine that the 3D
> >structure of a biological sample can be very different for different 
> >locations. In cells for example, the nuclear area is thicker 
> compared
> >to the cytoplasmic area towards the edges. So one can
> imagine that the
> >axial diffusion contribution to the recovery curve will be different
> >for both regions. Again, this is also an issue for some of 
> the physical
> >FRAP models.
> >
> >So in conclusion, although it should be possible for the empirical
> >method to work, I think it may be challenging to get correct 
> >measurements. But you seem to get good results with it, 
> which is great
> >of course. Can I ask, did you ever try to apply the method to a
> >viscosity series of the same probe and check whether you can 
> obtain the
> >Stokes-Einstein relation (D propotional to the reciprocal viscosity)?
> >
> >Best regards,
> >
> >Kevin
> >
> >-----Oorspronkelijk bericht-----
> >Van: Confocal Microscopy List [mailto:[log in to unmask]]
> >Namens Reece, Jeff (NIH/NIEHS)
> >Verzonden: woensdag 24 november 2004 16:29
> >Aan: [log in to unmask]
> >Onderwerp: Re: FRAP experiments
> >
> >
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >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
> >[log in to unmask]
> >
> >
> >>  -----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
> >>
> 
> 
> -- 
>                ****************************************
> Prof. James B. Pawley,                           Ph.  608-263-3147 
> Room 223, Zoology Research Building,               FAX  608-265-5315
> 1117 Johnson Ave., Madison, WI, 53706  [log in to unmask]
> "A scientist is not one who can answer questions but one who
> can question answers."  Theodore Schick Jr., Skeptical 
> Enquirer, 21-2:39
>