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Obviously it is possible to do any amount of damage with excessive
laser intensity. That is the basis of laser microdissection, after
all. But one careless experiment doesn't invalidate a technique.
Others (and I refer particularly to the work by Julia Hush and
Peter Hepler, which I've posted here before) were able to see
recovery in bleached areas of microtubules. Cells had been
microinjected with labelled tubulin.
There are unlimited methods of doing an experiment wrongly, but
usually a much smaller number of ways to do it properly ...
Guy
Jim Pawley wrote:
> 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
>
--
Associate Professor Guy Cox
Electron Microscope Unit,
University of Sydney,
NSW 2006, Australia
Phone:+61 2 9351 3176 Fax:+61 2 9351 7682
http://www.guycox.net
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