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Hello,
A friend of mine recently made me aware of a newly published paper that delivers a fairly comprehensive analysis of many different organic dyes that are suitable for super-resolution localization microscopy:
http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.1768.html
Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging
• Graham T Dempsey
• Joshua C Vaughan
• Kok Hao Chen
• Mark Bates
• Xiaowei Zhuang
Abstract:
One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low–cross-talk, four-color super-resolution imaging.
Haven't had a chance to read it yet, but there's lots of valuable information here. I seem to recall there many have been a similar review that looked at the differences between the various photo-activatable / photo-switchable fluorescent proteins as well. The only one that comes to mind right now is this one:
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2011.03505.x/abstract
Highlights of the optical highlighter fluorescent proteins
• G. H. Patterson
Abstract:
The development of super-resolution microscopy techniques using molecular localization, such as photoactivated localization microscopy, fluorescence photoactivated localization microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy with independent running acquisition and many others, has heightened interest in molecules that will be grouped here into a category referred to as ‘optical highlighter’ fluorescent proteins. This review will survey many of the advances in development of fluorescent proteins for optically highlighting sub-populations of fluorescently labelled molecules.
To me it seems that there are new probes for super-resolution imaging being developed and described in the literature almost monthly now. It's a bit hard to keep up with all that. What might be the best and brightest probe today might not be so in a year's time. We live in exciting times, I guess.
Cheers,
John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca
On 2011-11-15, at 8:14 AM, George McNamara wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> rsEGFP (1,000 cycles)
> http://www.abberior.com/index.php?
> option=com_content&view=article&id=96&Itemid=92
>
> Excitation: 491 nm
> Photoactivation: 405 nm
> Photodeactivation: 491 nm
>
> Usable in PALm/STORM/FPALM precision localization or STED-like imaging
> formats.
>
> note: 488 nm would work fine. I suspect that a 3D-EasySTED ("Easy-rsEGFP-
> nanoscope") segmented waveplate optimized for 1/2 Lambda at 488 nm would
> enable 488 nm depletion doughnut with 458 or 476 nm providing close-enough-
> to-Gaussian excitation. That is, if the 3D-EasySTED SWP could be added to a
> confocal microscope that has 405, 458/476, and 488 nm laser lines, could turn
> the existing confocal's into 40 nm XY, and from Matthias Reuss's PhD
> dissertation ~200 nm Z-resolution. See my previous post on 3D-EasySTED for
> references.
>
> The Grotogohann et al Nature paper did not cite Bogdanov et al (Evrogen's
> DMEMgfp) or Matsuda ... Sedat paper on riboflavin's impact on EGFP
> photostability, so high vs low riboflavin might be an additional way to modulate
> rsEGFP performance. Low riboflavin would also reduce "autofluorescence"
> background.
>
> ***
> Another recent Hell lab paper is on:
>
> Dreiklang (150 cycles, note three wavelengths in play)
>
> Dreiklang is a reversibly switchable fluorescent protein (RSFP) with a high
> fluorescence quantum yield and a large extinction coefficient. In contrast to
> all other known RSFPs today, Dreiklang has the unique property that besides
> two wavelengths used for switching between two different modifications –
> fluorescent and dark – a third wavelength can be used independently to read
> out the fluorescence. As a consequence, the fluorescence readout and
> photoswitching are completely decoupled processes.
>
>
> Excitation: 515 nm
> Photoactivation: 405 nm
> Photodeactivation: 365 nm
>
> note: too bad my LSM510's Enterprise I laser (351/364nm) died (in January ...
> I am about to ship it off to Laser Innovations on consignment if anyone wants
> to pay L.I. to refurbish it for your lab). Hoefully Stefan Hell and colleagues (or
> someone on the listserv) will report a high performance Ti:Sapphire laser
> wavelength to perform the photodeactivation well.
>
>
> Publications were recently in Nature and Nature Biotechnology (October
> issue). NBt also had a commentary by Joshua Vaughan and Xiaowei Zhuang on
> the two papers.
>
> On Tue, 4 Oct 2011 11:29:37 +0100, Julia Edgar
> <[log in to unmask]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All
>> Can anybody recommend a good photo-switchable and/or photo-activatable
> reporter.
>> Thank you
>> Julia Edgar
>> University of Glasgow
>>
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