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Mario,

to my knowledge, there isn't a lot of information on the topic of sample
heating during the photobleaching phase of a FRAP experiment. Only this:

1. Lopez et al. (see ref. below) used fluorescein as a pH indicator to
measure the temperature-induced pH change of a Tris buffer solution around
its pK. Under their experimental conditions (epi fluorescence microscope
with 100W mercury arc lamp, spot radius of about 5 micron, about 500 ms
illumination time), they measured a temperature increase of less than 0.4 K.

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.

2. In a publication of our own lab (De Smedt et al; ref. see below), the
influence of the laser beam on the temperature of the sample was examined by
a theoretical calculation. For details see:

De Smedt, S. C., A. Lauwers, J. Demeester, Y. Engelborghs, G. Demey, and M.
Du. 1994. Structural information on hyaluronic acid solutions as studied by
probe diffusion experiments. Macromolecules 27:141-146.

In short, the results of the calculations are that, assuming an illumination
power of 1 mW focused in a (focal) volume of (10 micron)^3, the temperature
increases slightly (about 0.15 K) during the first 5 ms after which the
temperature remains almost constant (steady state between heat conduction
and absorbed laser energy). When the bleaching beam is switched off, the
temperature falls back to its original level within about 5 ms. A watery
solution was assumed in the calculations.


In both cases the increase in temperature is very small (order of 0.1 K).
That is why, in general, a rise in temperature during bleaching is not
thought to be an issue.

Best regards,

Kevin

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List
> [mailto:[log in to unmask]]Namens Mario M. Moronne
> Verzonden: vrijdag 10 oktober 2003 20:11
> Aan: [log in to unmask]
> Onderwerp: Re: FRAP
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Scott,
>
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Here is a question I have been wanting to bring up.  Is a reaction with
> >triplet oxygen the only way to quench an XFP?
>
> Do you mean quenching or bleaching? Actually, both have the same
> answer: triplet oxygen is not the only way to get either effect. You
> probably mean bleaching, but just to mention it, iodide and
> nitroxides (spin labels) are useful fluorescence quenchers.
>
> As for bleaching, that is a very complex bit of photochemistry and
> still an area of research. Because oxygen is at about 200 uM in
> solution and 2 mM in membranes, it represents a highly mobile and
> reactive solution/membrane component unless specifically removed by
> scavengers. Singlet oxygen is highly reactive leading to superoxide,
> H2O2, e-, H and OH end products. Practically any of these can cause
> conjugated ring cleavage and killing of a fluorescent probe. In
> addition, an excited state fluorophore can undergo internal bond
> rearrangement (what you are calling denaturation?). There is always
> some finite probability for this to occur that will vary from
> fluorophore to fluorophore. One might think of this as the inability
> of the probe to dissipate internal "heat" before decomposition takes
> place.
>
> Heat dissipated in solution is not likely to be a factor except in
> structures or environments that have poor heat diffusion constants.
> However to be sure that this not important to your particular
> application, one must test it. I found it quite amusing (and a bit
> disconcerting) to discover that with ordinary 100 Watt
> epi-illumination, I could melt and refreeze small organic crystals
> with a melting point of 64 deg. C using a 0.75 NA 20x objective
> simply by opening or closing my illuminator's iris. Think of what a
> 1.4 NA lens might do with laser power densities, even in a high heat
> capacity-rapid diffusion medium such as water. Someone who is in the
> FRAP business might want to comment on the power and heating issues
> and good controls in this regard.
>
>
> >I know that for the excited
> >state to cross over to a state where it can fluoresce, it must release
> >energy as heat.
>
> Don't think that this is really true. It is just that absorption
> takes about 1E-15 secs., vibrational transitions to complete about
> 1E-12 secs., and >= 1E-9 sec for emission for most fluorophores. The
> biggest factor contributing to the broadness of the emission (and
> excitation) spectra are the vibronic energy levels occupied by the
> probe. And, of course, this gives that all important Stokes shift we
> need for label discrimination.
>
> >Can it release enough heat to denature itself and possibly
> >proteins around it?
>
> See above for first part, but if bond rearrangement occurs, a
> reactive form of the probe could crosslink with a nearby protein.
>
>
> >I know with FRAP many of the photobleachings tend to
> >use a ton of laser power to ensure complete bleaching.  This
> seems like it
> >could lead to problems with thermal denaturation.  It could also
> lead to a
> >discrepancy in results if one person pours a lot more laser
> power into their
> >bleach area than another person does.
>
> Again, someone who knows better than I, can better explain controls
> that would seem to be necessary. Your concerns appear to be valid.
> Any takers?
> --
> _________________________________________________________________
> Mario M. Moronne, Ph.D.
> NanoMed Technologies LLC
> President and CTO
> ph (510) 528-2400
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