Standards for SIM resolution

[Joshua Zachary Rappoport]

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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

[Alison J. North]

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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

[Indig, Fred (NIH/NIA/IRP) [E]]

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Alison, how is this better than sub-resolution beads?

Sincerely,
Fred


Fred E. Indig, Ph.D.
Head, Confocal Imaging Facility

Biomedical Research Center (BRC) Rm 8B135
National Institute on Aging/NIH
251 Bayview Blvd.
Baltimore, MD  21224-6825

Tel. [phone]
Fax  [phone]
[hidden email] 

________________________________________
From: Alison North [[hidden email]]
Sent: Tuesday, March 01, 2016 3:39 PM
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

[Sylvie Le Guyader]

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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
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone] New number!
LCI website

-----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

*****
To join, leave or search the confocal microscopy listserv, go to:
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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

[Ann-W]

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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





--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


-----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
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone] New number!
LCI website

-----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

*****
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 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

[Alison J. North]

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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:

> *****
> 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

[Pablo Hernandez-Varas]

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*****

Dear Ann and all,

We have, here at Nikon, also tested the nanorulers from Gattaquant with good results.
However, their stability is a problem.

Argolight is a small French start-up also producing standard samples for confocal, and they
are now designing a prototype for SIM. Here a link to their webpage:

http://argolight.com/

We have tested it and the sample contains many interesting features only resolvable by
SIM. We were very happy with the sample, since it is robust, long-lasting and very stable
(no bleaching). Please contact them for more information, and you may possibly be able to
provide feedback to them since they are finalizing the design phase. Unfortunately, the
sample is not commercially available yet, to my knowledge.

I hope this helps. Kind regards,
Pablo.

[Julio MATEOS_LANGERAK]

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Dear all,

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.

Cheers, Julio

> 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