Posted by
Tim Feinstein-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580175.html
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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/S0006349508000763Markus 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:
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> 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