Confocal Microscopy List - Re: Pulse compression and in vivo imaging

Re: Pulse compression and in vivo imaging

Posted by Wolfgang Staroske on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Pulse-compression-and-in-vivo-imaging-tp6557894p6594131.html

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Hi George,

thanks for your literature. I checked both, but concentrated more on
Arnolds PhD thesis. (I know him personally, as he works now as CW-STED
developer with Leica, and we had such a demo here, with a rather
problematic start)

In the paper they don't say anything about the electronic states behind.
Arnold suggests that the depopulation goes T1 -> Tn -> Sn -> S1 -> S0.
The same mechanism which I suggested for the bleaching (T1 -> Tn ->
dead). In the end I think it is intensity dependent, because the rate
for ISC from Tn to Sn competes against photon driven ionization from Tn.
There is a nice graph in his thesis, where it is visible that there is
an optimal T-depopulation laser power, and with higher laser powers the
fluorescence decreases again, I think due to photobleaching. But in
2-Photon-excitation you have no control on that laser power, because it
is your pulsed IR laser (Arnold had 300mW cw @ 671nm, our 2-Photon-laser
has 300mW averaged in several hundred fs @ 80MHz - I think a rather
common system). Additionally he clearly states, that this method
doesn't work at room temperature and T-REX (Donnert et al., PNAS 103,
p10440) has to be used, which is in principle only waiting. So I still
would bet for bleaching at room temperature.

Of course the only way to figure that out, is to measure it in a pulsed
regime as in Arnolds thesis. First excite the dye of interest with a
pulsed one-photon laser and record the fluorescence. Then do the same
but use your two-photon pulse after some ns (fluorescene should be
decayed) as a second pulse for T-depletion or bleaching, and compare
both traces. Of course in second run, record only the fluorescence from
the one-photon excitation. T-Depletion should increase the fluorescence
signal, while photobleaching should decrease it.

Bye Wolfgang

Am 20:59, schrieb George McNamara:

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> 
> Hi Wolfgang,
> 
> Check out:
> 
> Synchronously amplified fluorescence image recovery (SAFIRe).
> </pubmed/19902923> Richards CI, Hsiang JC, Dickson RM. J Phys
> Chem B.
> 2010 Jan 14;114(1):660-5. PMID: 219902923 (dyes that form triplet state
> readily at room temp.). Also cites some earlier work and they have a
> later FRET-SAFIRe paper.
> 
> and
> 
> Arnold Giske's cryoSTED PhD dissertation
> 
> http://archiv.ub.uni-heidelberg.de/volltextserver/volltexte/2008/7969/pdf/CryoSTED_microscopy_PhDThesis_agiske.pdf
> (if the pdf comes up blank initially, refresh the web page and/or
> scroll
> down).
> 
> Dr. Giske mostly used ATTO 532 (which has low rate into triplet
> state(s)
> at room temp), 671 nm for triplet depletion (Gaussian spot superimposed
> on confocal excitation spot), and as the title indicates, cold.
> 
> I am wondering whether triplet state depletion is worth mentioning
> as a
> potential opportunity to improve signal intensity for our STED proposal
> (if we have the right laser). My current limited understanding is that
> very good fluorophores will not benefit much compared to yet another
> variable to confuse reviewers and (if we get the money) users.
> 
> Enjoy,
> 
> George
> 
> 
> -------- Original Message --------
> Subject:     Re: Fwd: Re: Pulse compression
> and in vivo imaging
> Date:     Fri, 15 Jul 2011 09:31:47 +0200
> From:     Wolfgang Staroske
> <[hidden email]>
> Reply-To:     Confocal Microscopy List
> <[hidden email]>
> To:     [hidden email]
> 
> 
> 
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> 
> Hi George,
> 
> that's a good point, I haven't thought about this possibility yet.
> 
> Destruction would mean excite the molecule into higher triplet states
> until its is ionized.
> Triplet state depletion would be something like stimulated emission
> without emission of a photon.
> 
> I would favor the first one, because this process is quantum
> mechanically allowed and the energies of the IR photons should be
> enough
> to go up the electronic states.
> For the triplet-state depletion, I think the probability is as low as
> for the inter-system-crossing from the singlet to the triplet
> state and
> additionally the energy of  the triplet state is probably
> higher than
> the energy of the IR photon. Of course that could be a
> two-photon-process with enough energy than in, which would probably
> happen not in the same pulse but in the next ones. On the other hand I
> never heard of triplet-state depletion in one-photon-excitation and if
> you get the same signal in one and two-photon excitation the
> probabilities of absorption / stimulated emission (of one or two
> photons
> respectively) should be the same.
> 
> But at all I'm not an expert in quantum mechanics, the only fact I can
> state is that in the case of two-photon excitation the triplet
> lifetime
> is either very short (below time resolution<250ns) due to
> triplet state
> depletion or infinite due to photobleaching.
> 
> Bye Wolfgang
> 
> 
> Am 20:59, schrieb George McNamara:
> <blockquote type=cite> *****
>   To join, leave or search the confocal microscopy
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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:
>   <blockquote type=cite>*****
>   To join, leave or search the confocal microscopy
> listserv, go to:
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>   *****
>   
>   Dear all,
>   
>   Am 20:59, schrieb James Pawley:
>   <blockquote type=cite>&lt;br&gt;JP:
> One more factor. As 2p is
>  pulsed, the duty cycle
>   &lt;br&gt;is usually less than 10%. This
> means that people
>   &lt;br&gt;often work nearer to
> singlet-state saturation
>   &lt;br&gt;when using 2photon (to get an
> image in the same
>   &lt;br&gt;scan time). This means that a
> lot more excited
>   &lt;br&gt;molecules are present in the
> very high excitation
>   &lt;br&gt;field near the centre of the
> focus, and increases
>   &lt;br&gt;the likelihood of "one-plus-one"
> (or maybe 2 plus
>   &lt;br&gt;one?) overexcitation. Many
> smart, 2-photon folks
>   &lt;br&gt;blame this for much of the increased
>   &lt;br&gt;bleaching/excitation noted.
>   </blockquote>
>   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
>   
>   
>   </blockquote>
>   
>   </body>
>  </html>
>  </html>
> </blockquote>
> </body>
> </html>
> </html>

--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

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

Tel.: +49 (0) 351 463-40316
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