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In STED, like in some other high resolution imaging techniques, were a 
smaller excitation spot is used to get a higher resolution, the result 
will be less photons coming out of the sample. So you either increase 
your laser power resulting in more bleaching or you increase your 
detection sensitivity by using the most sensitive detectors. With such 
low amount of photons you off course never get very high signal to noise 
ratio's. Sadly enough you can't get it all.

Gert

on 9-11-2007 4:11 Ella Tour said the following:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Martin,
>
> I saw the STED demo yesterday in San Diego as well. I was also very 
> impressed by the gain in resolution of the histone staining sample 
> detected with STED comparing to the normal confocal mode of this Leica 
> SP5 machine. As you said, photobleaching might be a problem, and the 
> STED image looked great only when detected with Avalanche Photodiode 
> Detector, and very dim with poor signal/noise ratio when detected with 
> PMT. We've tried to image our sample stained with Alexa 647 dye, but 
> it did not work with STED.
>
> For STED imaging, the Leica people recommended using 
> 2,2'-Thiodiethanol (Fluka, 88559), the new mounting medium described 
> in the following paper from Stefan Hell lab:
>
> Staudt, Lang, Medda, Engelhardt and Hell (2007) Microscopy Research 
> Technique 70:1-9.
>
> Best wishes,
>
>> Ella
>
>
>
>
>
>> 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
>
>

-- 
wigGert van Cappellen, [log in to unmask]
TEL +31-10-70 43578; FAX +31-10-7044736
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