Re: Standards for SIM resolution

Posted by Ann-W on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Standards-for-SIM-resolution-tp7584830p7584841.html

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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
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: 02 March 2016 09:01
To: [hidden email]
Subject: Re: Standards for SIM resolution

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Hi Alison

Just out of curiosity, why did you choose the 80RG and not the 20nm one since you wanted to calibrate STORM? Or was the 80 was only your best second choice since the STORM nanorulers did not work?

Med vänlig hälsning / Best regards
 
Sylvie
 
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alison North
Sent: den 1 mars 2016 21:39
To: [hidden email]
Subject: Re: Standards for SIM resolution

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Hi Josh,

We have used both the SIM nanorulers to check our OMX and the 80RG PAINT nanorulers to check our STORM system.  Both work well though they can be a bit tricky to find at first.  We have had less luck yet with the STORM nanorulers in solution, so I would be interested to hear if anybody has worked out the kinks for that sample!
Give me a call if you want to talk about it,

Best,
Alison

On 3/1/2016 2:42 PM, Joshua Zachary Rappoport 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.
> *****
>
> Does anyone have a good recommended standard for SIM resolution?
> Has anyone tried these?
> http://www.gattaquant.com/products/gatta-sim-nanoruler.html
>
> Thanks!
>
> Josh