Posted by
Alison J. North on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580180.html
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Dear George,
You are absolutely correct that the refractive index of the immersion
oil is critical for 3D-SIM, which is why I feel I have to respond to
your posting and expand it a bit, in case any new or potential OMX users
out there get confused. You say that the 1.512 oil was "the best oil" -
I think it needs to be clarified that you mean this was the best oil for
that particular sample, looking at the particular wavelength
fluorochromes you were imaging, and working at the exact temperature of
that room. In picking the oil for any OMX experiment you need to first
check the room temp (if only ours were perfectly stable that wouldn't be
an issue, but sadly we do get 1 degree fluctuations from day to day
depending on the weather etc., despite having stipulated that our
engineers needed to give me the most stable room environment
possible!). Then you need to consider the sample prep - by encouraging
people to use the high performance coverslips and to try to stick to
ProLong Gold (talking about fixed samples only of course) we aim to
minimize the amount of time we need to optimize the oil r.i. each time.
But you will still need to decide which is the most critical channel in
your experiment - if you are most interested in the red signal then you
will pick a different oil than if the blue or green signals are the most
critical, as you won't get a perfectly optimal signal in every channel
with just one oil.
I also think you are being a tad unfair on SoftWorx - I can't say I find
it at all painful to use. Every software has its glitches and annoying
features but of the many different softwares we have in our facility we
actually find SoftWorx to be one of the most user-friendly - and it
hardly ever crashes, which is a big plus in my book. I don't know which
tedious manual steps you are referring to, but the more I have worked
with the system, the more I want to slow the users down and force them
to look at each log file while the reconstruction is going along.
Checking numbers in the logs like the line spacing, the amplitude, and
the ko angles gives a good initial indication of whether you have a
decent data set or not. If our users just ran everything as automatic
batch files I fear that the number of artifacts published would shoot up.
Anyway, that's just my two cents and I expect you will disagree
vehemently! :-)
All the best,
Alison
>
> 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.
>
>
--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab ++ 212 327 7486
Fax: ++ 212 327 7489