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am dyin leave me alone11At 15:21 11/29/04 +0000, you wrote:
>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
>>
>
________________________________________________________-


Daniel Mackay, B.Sc., FRMS, AIBMS
Advanced Microscopy Unit/Drosophila Laboratory
Section of Old Age Psychiatry/Department of Neuroscience
Institute of Psychiatry
De Crespigny Park
Denmark Hill
London
SE5 8AF
0207 848 0553 - office
0207 848 0554 - Microscopy Suite
0207 848 0119 - Drosophila Lab.

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