Posted by
Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-imaging-with-multiphoton-confocal-tp6297623p6319819.html
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Hi Kate
Do you really need/want two Tl:S lasers for this? It may be better to
use a visible and a Ti:S as I can foresee problems with continued
photolysis of the cage with the probe beam if it's 2P... In our work
measuring diffusion via 2P we used an Ar-ion with a 2P photolysis
spot that could be either stationary or moved with the probe using the
same scanning system. For analysis the stationary spot (produced by
our own custom optics coupling the Ti:S bean to the rear aperture
independent of the scanner) was the best way to go. The trouble is not
all microscope bodies can give you the room to a fit in a short pass
dichroic to do this. In our old Axiovert, we had to machine up a new
coupling component to take a small dichroic on a slider. A diagram of
the layout is here:
Soeller, C. & Cannell, M.B. (1999) Two-photon microscopy: Imaging in
scattering samples and three-dimensionally resolved flash photolysis.
Microsc. Res. Tech. 47:182-195.
Cheers Mark
On 1/05/2011, at 1:06 AM, Kate Luby-Phelps wrote:
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>
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>
> So far we have been using one laser, which means we miss the
> beginning of the
> timecourse after photoactivation because it takes several seconds
> for the Ti:S to
> tune and mode-lock. So the experiments where 2P photoactivation has
> been
> useful involve diffusion through gap junctions between cells in a
> cluster or chain
> because the spread of the photoactivated dye between cells is
> relatively slow.
> However, in June we will take delivery on a new 2P system with two
> Ti:S lasers
> so we can do it simultaneously at two different wavelengths.
>
> Kate