Re: what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?

Posted by Douglas Richardson on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580176.html

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This is STORM.  It doesn't work with every dye, but many.  See here:

Evaluation of fluorophores for optimal performance in localization-based
super-resolution
imaging<http://www.nature.com/nmeth/journal/v8/n12/full/nmeth.1768.html>

   - Graham T Dempsey<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-1>
   ,
   - Joshua C Vaughan<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-2>
   ,
   - Kok Hao Chen<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-3>
   ,
   - Mark Bates<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-4>
   - & Xiaowei Zhuang<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-5>

Nature Methods 8,1027–1036(2011)doi:10.1038/nmeth.1768

-Doug



On Thu, Apr 11, 2013 at 10:20 AM, Tim Feinstein <[hidden email]> wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
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> *****
>
> I wonder whether anyone has experience with the inducible dark states
> approach reported by Christian Soeller in New Zealand.  In theory one could
> do super-resolution TIRF with any fluorophore you want.
>
> http://www.sciencedirect.com/science/article/pii/S0006349508000763
>
> Markus Sauer followed up with a protocol:
>
> http://www.nature.com/nprot/journal/v6/n7/abs/nprot.2011.336.html
>
> Has anyone tried this?
>
> cheers,
>
>
> TF
>
> Timothy Feinstein, PhD
> Visiting Research Associate
> Laboratory for GPCR Biology
> Dept. of Pharmacology & Chemical Biology
> University of Pittsburgh, School of Medicine
> BST W1301, 200 Lothrop St.
> Pittsburgh, PA  15261
>
> On Apr 11, 2013, at 8:35 AM, Tom Blanpied wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > We use mEos whenever possible. mEos2 seemed great to us, as we never saw
> > any effects of the small dimerization tendency. mEos3 does look to be at
> least
> > as good, and it does seem a bit brighter; it is our go-to probe now. We
> avoid
> > the bulk of the tdEos, but did think it was a good molecule.
> >
> > PA-mCherry we thought was pretty terrific at first, but we no longer use
> it
> > unless we really need the separate GFP channel for something. It is
> > considerably dimmer in our hands than mEos  (photons/frame and number of
> > frames till bleaching), and also seems to have a tendency to produce a
> smaller
> > population of photoactivated molecules. We've not tried to determine
> whether
> > this was due to expression level or folding or a non-activatable
> fraction, but it
> > does not seem to be due simply to the diminished ability to localize dim
> or
> > quickly bleached molecules.
> >
> > PA-GFP and Dronpa we did a little bit with, but in general we have so
> much
> > autofluorescent green stuff in our relatively old cultured neurons that
> anything
> > in that spectral range will be unsatisfying. We finally managed to
> obtain some
> > PS-CFP2, but haven't yet measured much about it; we're hopeful it will
> be the
> > best of that range.
> >
> > I'll be curious to hear comparisons of dendra2-type molecules.
> >
> > Tom
>