GSD (dSTORM) help

[Verónica Labrador]

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Dear listers,

     My name is Veronica Labrador and I work as a microscopy technician at Centro Nacional de Investigaciones Cardiovasculares (CNIC), in Madrid (Spain). Among the different systems available we have a Leica 2D GSD system.

     One of  our users, Marta Portela, is currently doing some dSTORM acquisitions. Single molecule localization microscopy is not one of the most requested techniques and we would like you helping us solving some problems/doubts.

     We are trying to study the distribution of Histone marks in the nucleus that do not show a well defined and constant pattern. As what we have to visualize is not a "known" structure we are worried about choosing the right settings. We have some basic questions but most of the time this information is difficult to be found in the Methods' section of a paper. We would like to know whether there is some standard procedure within the field to approach these questions:

     -Which would be the optimal detection ratio (blinks/frame)?
     -How do we decide for how long we should image?
     -How do we choose the correct antibody concentration to get good results and not miss anything?

    On the other hand, we observe a lot of variation in the blinking performance between days. Unfortunately it is not suitable for us to use the liquid and most conventional buffers that help to maintain a robust performance: our sample itself has some volume and could move/rotate during acquisition in a liquid environment. After trying different mounting methods in order to avoid any movement we end up using a STORM buffer composed by a mix of MEA and glycerol, but we keep having this day-to-day variation. Is there a way to reduce variability between acquisitions?

Any help will be welcome!

Greetings from Madrid,

Marta and Veronica

[Michelle Peckham]

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Dear Marta and Veronica

We do dSTORM using a custom built 3D STORM set-up.

Some useful resources (there are probably a lot more - but here are a few)

https://www.nature.com/articles/s41592-020-0918-5 

is a good place to start perhaps (not GDStorm, but STORM) but will give you some idea about blinks per frame.

Also https://www.nature.com/articles/nprot.2011.336


https://www.esric.org
runs a course on super-resolution imaging - also a good place to go to to ask

Nikon Storm protocol https://www.research.uky.edu/uploads/nikon-storm-manual
Also has some useful information

Optimal detection ratio - hard to specify - not too few blinks - (as you will need to record for a long time to get enough localisations for your reconstruction), but not too many - so you have the problem of overlapping blinks, - it's a bit 'trial and error'. We do this by 'eye' (from long experience) - but there are probably papers out there where people have measured and have recommendations for the optimum rate.

How long to image - see resources above - again, until you've processed the data, it is hard to be specific - you need enough frames/localisations to be able to reconstruct your image, and remember blink rate decreases over time.  We typically use around 8000 frames (but it depends)

Correct antibody concentration (see above resources) - typically lower than in standard IF (maybe start with 5-10 fold lower)
But also depends on whether using directly labelled primary antibodies, or combination of primary and secondary antibodies.


All best

Michelle


On 26/08/2020, 16:34, "Confocal Microscopy List on behalf of Verónica Labrador" <[hidden email] on behalf of [hidden email]> wrote:

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    *****
   
    Dear listers,
   
         My name is Veronica Labrador and I work as a microscopy technician at Centro Nacional de Investigaciones Cardiovasculares (CNIC), in Madrid (Spain). Among the different systems available we have a Leica 2D GSD system.
   
         One of  our users, Marta Portela, is currently doing some dSTORM acquisitions. Single molecule localization microscopy is not one of the most requested techniques and we would like you helping us solving some problems/doubts.
   
         We are trying to study the distribution of Histone marks in the nucleus that do not show a well defined and constant pattern. As what we have to visualize is not a "known" structure we are worried about choosing the right settings. We have some basic questions but most of the time this information is difficult to be found in the Methods' section of a paper. We would like to know whether there is some standard procedure within the field to approach these questions:
   
         -Which would be the optimal detection ratio (blinks/frame)?
         -How do we decide for how long we should image?
         -How do we choose the correct antibody concentration to get good results and not miss anything?
   
        On the other hand, we observe a lot of variation in the blinking performance between days. Unfortunately it is not suitable for us to use the liquid and most conventional buffers that help to maintain a robust performance: our sample itself has some volume and could move/rotate during acquisition in a liquid environment. After trying different mounting methods in order to avoid any movement we end up using a STORM buffer composed by a mix of MEA and glycerol, but we keep having this day-to-day variation. Is there a way to reduce variability between acquisitions?
   
    Any help will be welcome!
   
    Greetings from Madrid,
   
    Marta and Veronica
   

[Michelle Peckham]

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And immediately I notice a typo!

80,000 frames! (not 8000)

On 26/08/2020, 17:09, "Confocal Microscopy List on behalf of Michelle Peckham" <[hidden email] on behalf of [hidden email]> wrote:

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    *****
   
    Dear Marta and Veronica
   
    We do dSTORM using a custom built 3D STORM set-up.
   
    Some useful resources (there are probably a lot more - but here are a few)
   
    https://www.nature.com/articles/s41592-020-0918-5 
   
    is a good place to start perhaps (not GDStorm, but STORM) but will give you some idea about blinks per frame.
   
    Also https://www.nature.com/articles/nprot.2011.336
   
   
    https://www.esric.org
    runs a course on super-resolution imaging - also a good place to go to to ask
   
    Nikon Storm protocol https://www.research.uky.edu/uploads/nikon-storm-manual
    Also has some useful information
   
    Optimal detection ratio - hard to specify - not too few blinks - (as you will need to record for a long time to get enough localisations for your reconstruction), but not too many - so you have the problem of overlapping blinks, - it's a bit 'trial and error'. We do this by 'eye' (from long experience) - but there are probably papers out there where people have measured and have recommendations for the optimum rate.
   
    How long to image - see resources above - again, until you've processed the data, it is hard to be specific - you need enough frames/localisations to be able to reconstruct your image, and remember blink rate decreases over time.  We typically use around 8000 frames (but it depends)
   
    Correct antibody concentration (see above resources) - typically lower than in standard IF (maybe start with 5-10 fold lower)
    But also depends on whether using directly labelled primary antibodies, or combination of primary and secondary antibodies.
   
   
    All best
   
    Michelle
   
   
    On 26/08/2020, 16:34, "Confocal Microscopy List on behalf of Verónica Labrador" <[hidden email] on behalf of [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.
        *****
       
        Dear listers,
       
             My name is Veronica Labrador and I work as a microscopy technician at Centro Nacional de Investigaciones Cardiovasculares (CNIC), in Madrid (Spain). Among the different systems available we have a Leica 2D GSD system.
       
             One of  our users, Marta Portela, is currently doing some dSTORM acquisitions. Single molecule localization microscopy is not one of the most requested techniques and we would like you helping us solving some problems/doubts.
       
             We are trying to study the distribution of Histone marks in the nucleus that do not show a well defined and constant pattern. As what we have to visualize is not a "known" structure we are worried about choosing the right settings. We have some basic questions but most of the time this information is difficult to be found in the Methods' section of a paper. We would like to know whether there is some standard procedure within the field to approach these questions:
       
             -Which would be the optimal detection ratio (blinks/frame)?
             -How do we decide for how long we should image?
             -How do we choose the correct antibody concentration to get good results and not miss anything?
       
            On the other hand, we observe a lot of variation in the blinking performance between days. Unfortunately it is not suitable for us to use the liquid and most conventional buffers that help to maintain a robust performance: our sample itself has some volume and could move/rotate during acquisition in a liquid environment. After trying different mounting methods in order to avoid any movement we end up using a STORM buffer composed by a mix of MEA and glycerol, but we keep having this day-to-day variation. Is there a way to reduce variability between acquisitions?
       
        Any help will be welcome!
       
        Greetings from Madrid,
       
        Marta and Veronica
       
   
   

[Ann-W]

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Hi Veronica

Also thanks Michelle, Veronica I'm very happy to chat with you about this. We do a lot of STORM with Histones (we would at the IGMM since we work very closely with the Bickmore and Gilbert groups).

I agree with Michelle's suggestions - I would strongly recommend use of AlexaFluor 647 as a label as this dye is one of the best characterised in the field and has a good duty ratio (blinks per frame). Particularly if it’s a structure which is unknown - using a dye which is very reliable for dSTORM will help. It probably makes most sense if you email me about the questions you have. I can also ask some of our Postdocs and Fellows who do work on STORM with Histones and other nuclear structures to comment. It is very tricky to set up and get consistent results.

Thanks

Ann

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-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Verónica Labrador
Sent: 26 August 2020 16:23
To: [hidden email]
Subject: GSD (dSTORM) help

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*****

Dear listers,

     My name is Veronica Labrador and I work as a microscopy technician at Centro Nacional de Investigaciones Cardiovasculares (CNIC), in Madrid (Spain). Among the different systems available we have a Leica 2D GSD system.

     One of  our users, Marta Portela, is currently doing some dSTORM acquisitions. Single molecule localization microscopy is not one of the most requested techniques and we would like you helping us solving some problems/doubts.

     We are trying to study the distribution of Histone marks in the nucleus that do not show a well defined and constant pattern. As what we have to visualize is not a "known" structure we are worried about choosing the right settings. We have some basic questions but most of the time this information is difficult to be found in the Methods' section of a paper. We would like to know whether there is some standard procedure within the field to approach these questions:

     -Which would be the optimal detection ratio (blinks/frame)?
     -How do we decide for how long we should image?
     -How do we choose the correct antibody concentration to get good results and not miss anything?

    On the other hand, we observe a lot of variation in the blinking performance between days. Unfortunately it is not suitable for us to use the liquid and most conventional buffers that help to maintain a robust performance: our sample itself has some volume and could move/rotate during acquisition in a liquid environment. After trying different mounting methods in order to avoid any movement we end up using a STORM buffer composed by a mix of MEA and glycerol, but we keep having this day-to-day variation. Is there a way to reduce variability between acquisitions?

Any help will be welcome!

Greetings from Madrid,

Marta and Veronica
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