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

Re: Pulse compression and in vivo imaging

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Pulse-compression-and-in-vivo-imaging-tp6557894p6586547.html

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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:

> *****
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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:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Dear all,
> 
> Am 20:59, schrieb James Pawley:
> <blockquote type=cite> JP: One more factor. As 2p is
> pulsed, the duty cycle
> is usually less than 10%. This means that people
> often work nearer to singlet-state saturation
> when using 2photon (to get an image in the same
> scan time). This means that a lot more excited
> molecules are present in the very high excitation
> field near the centre of the focus, and increases
> the likelihood of "one-plus-one" (or maybe 2 plus
> one?) overexcitation. Many smart, 2-photon folks
> blame this for much of the increased
> 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>

--
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
Fax.: +49 (0) 351 463-40342
E-Mail: [hidden email]
Webpage: www.biotec.tu-dresden.de