Re: Fwd: Re: Pulse compression and in vivo imaging

Posted by Wolfgang Staroske on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fwd-Re-Pulse-compression-and-in-vivo-imaging-tp6577135p6583516.html

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Dear all,

Am 20:59, schrieb James Pawley:

> <br>JP: One more factor. As 2p is pulsed, the duty cycle
> <br>is usually less than 10%. This means that people
> <br>often work nearer to singlet-state saturation
> <br>when using 2photon (to get an image in the same
> <br>scan time). This means that a lot more excited
> <br>molecules are present in the very high excitation
> <br>field near the centre of the focus, and increases
> <br>the likelihood of "one-plus-one" (or maybe 2 plus
> <br>one?) overexcitation. Many smart, 2-photon folks
> <br>blame this for much of the increased
> <br>bleaching/excitation noted.

I would like to comment on this. In FCS Experiments we see that all
dyes, even the ones which show a strong triplet fluctuation in
one-photon excitation, show no triplet fluctuation in the case of
two-photon excitation.

Our hypothesis for that is the following. The lifetime of the triplet
state is long enough, that each molecule, which entered the triplet
state, absorbs a third IR photon, which destroys the dye molecule. So
molecules which entered the triplet state are dark from this time point on.

In imaging of course this possibility is reduced because the laser is
scanned and pixel dwell times are usually in the range or below the
triplet state lifetime (few µs), while in FCS the residence time of even
small molecules are at least 20µs.

Bye Wolfgang


--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

Technische Universität Dresden
Biotechnology Center
Tatzberg 47/49
01307 Dresden, Germany

Tel.: [phone]
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Webpage: www.biotec.tu-dresden.de