Re: photo-switchable reporter .. rsEGFP (10000 cycles), Dreiklang (150 cycles)

[George McNamara]

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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
<[hidden email]> 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
>

[John Oreopoulos]

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To join, leave or search the confocal microscopy listserv, go to:
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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
> <[hidden email]> 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
>>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Og more immediate use with respect to ucrrent fluorescence microscopy
and photostable nanoscopy may be the Altman et al article - check out
the supplemental videos at

http://www.nature.com/nmeth/journal/vaop/ncurrent/abs/nmeth.1774.html#supplementary-information

The same AOP
http://www.nature.com/nmeth/journal/vaop/ncurrent/index.html has a fast,
live cell, 3D-SIM paper from Shao et al, fluorescent mechanical tension
sensor paper by Stabley et al., and deep light sheet 3D nanoscopy paper
by Zanacchi et al. I am also looking forward to reading the protein
stoichiometry paper by Kleinlogel et al.

Going back to STORM - has Nikon and its reagent partner(s?) announced
availability of "N-STORM dyes"?

George
p.s. while looking for press releases on N-STORM dyes, the most
interesting web page I came across was

http://www.brightdyes.com/FAQ.html#5



On 11/17/2011 2:18 PM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>> <[hidden email]>  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
>>>
>>>        
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami