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Stefan Hell recently published STED with continuous wave beams 
(Willig et al Nat Methods. 2007 Nov;4(11):915-8), so the beam shaping 
and co-alignment of the depletion beam may end up being more 
important than having a pulsed laser, and thus price might go down.

I am disappointed to hear that Z-resolution is not significantly 
improved. Did Leica neglect to put depletion "bars" in the system? 
Sounds like they were effective only in the XY "doughnut".

A lot of interesting papers have been published in 2007 on various 
methods for fluorescence nanoscopy. STED is by no means the only way 
to get to 100x smaller voxels than conventional confocal, multiphoton and TIRF.

STED Question: what happens to the fluorophore's excited energy when 
depletion occurs? Does the molecule return to the ground state (a) 
without emitting a photon or (b) emits a photon of the same 
wavelength (and direction? as in a laser) as the depletion photon?



At 02:22 PM 11/8/2007, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hey folks!
>
>I just got back from the Society for Neuroscience meeting in San 
>Diego, where Leica was giving a demo of it's Stimulated Emission 
>Depletion (STED) instrument.
>
>Seeing it was an interesting experience.  The microscope appeared to 
>improve resolution of some specimens (specifically, histones) by a 
>factor of probably 5-fold--there was a very pronounced improvement 
>and unless their scale bar was lying, they seemed to be down into 
>the 50 nm range.  It did not seem to offer as much improvement on 
>their muscle specimen, and photobleaching was a serious problem on 
>that one as well when they went up to high zooms.
>
>The STED module works only for one color (far red) and does not work 
>well with all fluorophores (--specifically, Cy5 apparently bleaches 
>too fast to be useful).  The fluorophores they recommended are the 
>ATTO 647 and 655 dyes.  Although STED provides an improvement in x-y 
>resolution, there's little or no improvement in the z-axis resolution.
>
>The instrument is essentially a Leica multiphoton microscope with 
>the STED unit as an attachment.  It can be used in single-photon, 
>multiphoton or STED modes.  Price is $1.3 million USD.
>
>I'd be interested in hearing from other folks who talked to Leica 
>about this machine and who saw one of the demos.  If I were buying 
>one of these items, it'd be worried about it suddenly becoming 
>obsolete (as happened with some of the early 2-photon instruments) 
>due to some new development in the pipeline.  Does anyone have any 
>sense of how likely that is?  I'd also be concerned about how suited 
>it is to all preparations--will it work only with the strongest 
>labeling?  Do the ATTO dyes require particular mounting media?
>
>Thanks in advance!
>
>Martin
>--
>Martin Wessendorf, Ph.D.                   office: (612) 626-0145
>Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
>University of Minnesota             Preferred FAX: (612) 624-8118
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
>Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu






George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
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Analytical Imaging Core Facility)