Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580179.html
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Hi Tom,
thanks! ... Michael Davidson, who told me: "The best photoswitchable
proteins are tdEos, mEos4, PS-CFP2, PA-mCherry1, and Dendra2."
I anticipate getting all of these from Michael (collaboration with our
lab - no redistribution from us, so anyone interested will need to
contact Michael directly for plasmids). Looks like I will start with mEos4.
Sincerely,
George
p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A
couple of observations (which may not be new to the listserv):
* refractive index of the immersion oil matters a lot. We (Tomasz and
Anna Zal were the trainers) had best results with one of Michael
Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus
100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils
were noticably worse in the reconstructions. The RI=1.518 was obviously
a wrong choice from the acquisition screen (no or low contrast fringes).
* Michael's DAPI in CytoSeal photoconverted to green fluorescence after
prolonged excitation with 405 nm laser (several of us have seen, and
mentioned on listserv, this with Hoechst ... I suppose it is possible
the slide was mislabeled and was actually Hoechst???).
* Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I.
changes from the edge to the middle of the coverglass. This makes sense
in that the stuff solidifies by outgasssing a [relatively low refractive
index] solvent. Since 0.002 steps in oil matter, it appears that the
Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge
to middle. This suggests that instead of spending time to change the oil
for different areas (distances), that it may be more efficient to select
an oil and then search the coverglass for optimum R.I. match (best fring
contrast and then final check on the reconstructed image). ... I may
decide to use Prolong Gold with "open face" imaging dishes, or buy and
test CytoSeal etc.
* The Applied Precision reconstruction software is called "SoftWoRx" but
a much more accurate name is "painfully tedious manual steps to do
everything anti-productivity WoRx".
* Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200
on Monday (Tomasz and Anna doing the training again). Maybe I'll end up
optimizing sample preps for this instead of the OMX.
On 4/11/2013 9:14 AM, Tom Blanpied wrote:
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>
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>
> Hi,
> Sorry, I conflated two molecules in my previous post.
> In our experience, PA-mCherry provides adequate numbers of molecules, though
> isn't as bright or long-lived as mEos2/3.
> PA-TagRFP is considerably brighter than PA-mCherry. However, on two or three
> different proteins with side-by-side comparisons, it has provided a smaller number
> of localized molecules than Eos or Cherry. Thus, longer tracks than PA-mCherry,
> but fewer.
> Tom
>
>
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