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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
>[log in to unmask]
Long term followers of this list will be disappointed if I don't
voice my traditional cautionary advice.
BEWARE OF FRAP!.
Light bright enough to bleach fluorescent molecules in "a short time"
is likely to severely disrupt cellular ultrastructure. In one
celebrated case, FRAP was used not to measure diffusion but to mark a
band of microtubules in a mitotic apparatus. The idea was to see if
the mts themselves were moving or if the chromosomes were moving
along them. The bleached bands did not move.
But the chromosomes moved anyway. Looked interesting until Richard
MacIntosh at U. Colorado performed the same experiment but followed
it up by imaging the same areas of exactly the same cells in the EM.
The stained mts had been destroyed. New mts then formed to move the
chromosomes.
Stung by this revelation the authors of the original study made a
preparation of isolated fluorescent mts on a patterned "finder"
coverslip. They bleached them at different power levels and then
looked at the mts in SEM. Generally speaking, bleaching destroyed
mts.
I am sorry that I cannot remember the exact power levels and
wavelenghts used in these experiments and it is possible that lower
damage levels may characterize the process when other dyes and
wavelengths are used.
However, I think that it is only safe to assume major damage is the
likely result UNTIL correlative EM studies show this is not the case.
We sometimes like to assume that cells are just like water-balloons
with nuclei and a few other organelles rolling around inside. High
Resolution FE-SEM studies of freeze-fractured cells show that they
are crammed with all sorts of generally ignored structures:
structures that may be destroyed by intense light, thereby changing
diffusion coefficients and perhaps even changing the "scene" into one
of directed flow to repair damage.
So be warned, any diffusion coefficients that you measure from FRAP
experiments should be treated with great caution. They may just
indicate diffusion in a bomb crater.
Happy Turkey Day!
Jim Pawley
--
****************************************
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
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