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Hi Wolfgang,

why do you think you are causing photodestruction, as opposed to triplet 
state depletion back to the ground state?

thanks,

George


On 7/14/2011 10:30 AM, Wolfgang Staroske wrote:
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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
>
>


-- 


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami