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

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

Dear,

With a confocal microscope you can heat-up your sample very well. We have tested
this by parking a laser beam to a cristal of parafenyleen-diamine (a photografic
developer, also used as antifading) and we could see that the edge of the
cristal melted. The melting point of parafenyleen is around 80°Celsius.

There also are calculations done on heating up samples during scanning in a
confocal set-up. The weak point in all these is the question: how much
light(=energy,=heat) is absorbed and converted to heat instead of fluorescence.

If we like a near linear relation between fluorescence en concentration we need
to have very low dye concentrations (absorbance les than 0.1). Under these
conditions heating up by absobance of light can not be a problem for a laser
system, but halogen light can realy generate a lot of heat. The difficulty is
however how to measure it. I once looked for dyes that are sensitive to heat but
in this set-up you should correct for melting of the dye it self. It is not
possible to accurately measure the temperature with a thermometer when the bulb
of the thermometer is larger than the volume in which you like to estimate the
temperature this is the limit. Thus theoretical calculations are the only way
out, however you than need to agree on a model about heat transfer and
discipation in yoyr samples.

Bye

Patrick Van Oostveldt

 "Ignatius, Mike" <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> This listserv has talked extensively about thermal problems as relating
> to focus drift and the like.
>
> However, has anyone wondered or better yet, systematically addressed the
> concern of any local thermal effects on cells?  I am not talking about
> photoxicity, but rather are we heating the cells in the illumination
> field to any appreciable degree?
>
> I suspect FCS folks have some comment here.  The local effect of heating
> on Brownian motion must be controlled for.  But I am more interested in
> standard live cell imaging format: e.g. a 60/1.4  oil objective, on an
> inverted scope, with a dapi excitation filter set delivering light from
> a standard Hg or Xenon bulb.  Is it possible to raise the heat from RT
> (26 degrees) to say, 43 degrees?  For clarity we are concerned with
> avoiding this, not producing it.
>
> Mike Ignatius
>
> Molecular Probes/Invitrogen
>


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