Posted by
Alison J. North on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Standards-for-SIM-resolution-tp7584830p7584844.html
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Hi all,
Sorry, I ran out of time to reply to Fred and Sylvie's questions
yesterday, plus, I am slightly afraid of getting sucked into a long
philosophical debate....
Fred, you asked why I prefer the nanorulers to sub-resolution beads.
Obviously I use beads all the time to check our systems - particularly
since there are still no commercially available 3D test samples (as far
as I know?) - and I have found it really important to check our
super-res systems in 3D as well as in 2D. However, I like the
GattaQuant nanorulers both for testing the systems and for teaching
purposes. We find that people get confused all the time about the true
meaning of resolution vs. precision and sensitivity. Obviously we can
measure the FWHM on the super-res systems just looking at beads or
microtubules - but when I'm trying to demonstrate resolution I find it
is more compelling to show the users a sample with the fluorescent
markers spaced at known distances from each other and show that we can
truly resolve them as being separate. With a monolayer of beads, you
don't know how far apart the beads actually are, you're just using the
microscope to work this out. In the past I have used antibodies
directed against known regions of the two parallel desmosomal plaques to
demonstrate this, but I am running out of those antibodies!
Sylvie, you asked why we purchased the 80 nm RG PAINT rather than the 20
nm ones. To be honest, I'd have liked to be able to order 40 nm or 60
nm ones, but those were not available. I was concerned that if we
ordered the 20 nm ones, these would be a bit too close to the resolution
limit for our STORM system and so if we had bad drift or bad algorithms
or whatever then we would never resolve the two spots. At least with
the 80 nm ones we can definitely resolve them, and we can check how good
our precision is etc. The main reason I wanted them is for users who
don't put enough time into sample prep and then complain that their poor
results must mean that our microscope isn't working. This way we can
shove on the PAINT slide and quickly show them that the system is
working just fine. It's sad that we have to do this, but sometimes we do.
I think that what GattaQuant is trying to do is a great first step. I
have already discussed with them that I would like them to add different
colours and resolutions. Personally, I would like them to make a slide
with 3 colours of SIM nanorulers - using 100 nm spacing in the
blue-emitting channel, 120 nm in the green, and 140 in the red. That
would match our OMX's resolution limits more closely than the currently
available ones so it would provide more stringent testing. Actually, I
would also like them to make some "co-localisation" nanorulers with the
different colours spaced at different resolutions, so that I could
finally have a standard slide to prove to our users that the higher the
resolution of the system, the less likely you are to see
co-localisation. It's amazing how this seems to be such a foreign
concept to so many of them, aaaargh!
The problem is, as Ann said, they do not last long and they also
photobleach, so you can't just pull them in and out of the freezer every
few months and expect to get the same results. Still, I am very glad to
see companies beginning to address our needs for better standardized
test samples.
All the best,
Alison
On 3/2/2016 11:39 AM, WHEELER Ann wrote:
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>
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> *****
>
> Similarly to Alison I used the Gattaquant Nanorulers as well and got great results for our Nikon-SIM. However we found that our rulers didn't last that long. I did ask for a 3 colour SIM nanoruler to be made so I could check resolution on the same sample (since we generally do multicolour on the SIM so the comparator for us was important).
>
> For the STORM I used the 40nm two colour sample, had a similar situation in as much as it doesn't keep that long but was useful. Its possible to get 40nm 640 beads - a previous thread a few weeks ago discussed this which I would use as an alternative to calibrate the STORM.
>
> Silvie, I would say here that the resolution the system gives very much depends on the algorithm used to reconstruct data and this is often dependant on the experiment done. Centroid fitting algorithms don't take into account several sources of false positives and negatives which can riddle STORM data - this is less of an issue with PALM since the Photoconvertable FPs blink only once. The merits and drawbacks of various algorithms are discussed in a nature methods paper from the 1st SMLM software benchmarking competition here:
http://www.nature.com/nmeth/journal/v12/n8/full/nmeth.3442.html>
> Best
>
> Ann
>
>
> Dr Ann Wheeler
> Advanced Imaging Resource, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU
> E:
[hidden email]
> T: 0131 651 8665
> W:
http://www.igmm.ac.uk/imaging.htm>
>
>
>
>
--
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