http://confocal-microscopy-list.275.s1.nabble.com/Standards-for-SIM-resolution-tp7584830p7584850.html
We are using a sample of the mentioned Argo-SIM slide from Argolight on our OMX.
Though the tests are preliminary, I think they are going to be great for maintenance purposes. They don't bleach (at least so they claim and indeed I didn't manage to) and you get a variety of patterns and motifs to test many things. They do not replace a good sub-resolution beads sample for some tasks and they are pricy, but the convenience pays off in routine checks in the context of an imaging core facility. Not only for SIM by the way.
Cannot give more detains as the slide is not yet released, but I would keep an eye on it. The principles are the same as with their current offer.
> On 02 Mar 2016, at 20:46, Alison North <
[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> 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:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> Post images on
http://www.imgur.com and include the link in your posting.
>> *****
>>
>> 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