Imaging for spatial transcriptomics platform

[Gaurav Joshi]

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Hi everyone,

Our facility is seeing an increase in the number of users bringing in
slides with tissues to image for 10X spatial transcriptomics platform.

We have been doing a tile and stitch for both H&E and IF based
platforms. We did not have any user issues with H&E based platform.
For the IF based platform, the user sees a degradation in their RNA.
We are using a Nikon crest spinning disk confocal for imaging, as it
does a great job at tile and stitch and gives speed. The user is
concerned that the laser might be degrading the RNA. The time that the
tissue stays outside during imaging is not of concern as the users
were able to get a good yield post H&E imaging during which the tissue
stayed outside for 90 minutes. With IF it's staying outside for
shorter than that. I do not have a reason to suspect that the laser is
degrading the RNA as there are a lot of variables with tissue
processing during IF based spatial transcriptomics.

In general, I want to know your experience with imaging samples for
spatial transcriptomics and any issues that you may have encountered
so that we can serve our users better.

Thank you,
Gaurav Joshi, PhD  |  Imaging Scientist
Integrated Cellular Imaging (ICI) core, Emory University
Health Sciences Research Building (HSRB)
EG72, 1760 Haygood Drive, Atlanta, GA 30322

[Sylvie Le Guyader]

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Hi Gaurav

What do you mean by 'degradation of their RNA'? Less dots? Aggregates? Not bright enough to be detected? Anything else?

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Gaurav joshi
Sent: 15 December 2020 00:10
To: [hidden email]
Subject: Imaging for spatial transcriptomics platform

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Hi everyone,

Our facility is seeing an increase in the number of users bringing in slides with tissues to image for 10X spatial transcriptomics platform.

We have been doing a tile and stitch for both H&E and IF based platforms. We did not have any user issues with H&E based platform.
For the IF based platform, the user sees a degradation in their RNA.
We are using a Nikon crest spinning disk confocal for imaging, as it does a great job at tile and stitch and gives speed. The user is concerned that the laser might be degrading the RNA. The time that the tissue stays outside during imaging is not of concern as the users were able to get a good yield post H&E imaging during which the tissue stayed outside for 90 minutes. With IF it's staying outside for shorter than that. I do not have a reason to suspect that the laser is degrading the RNA as there are a lot of variables with tissue processing during IF based spatial transcriptomics.

In general, I want to know your experience with imaging samples for spatial transcriptomics and any issues that you may have encountered so that we can serve our users better.

Thank you,
Gaurav Joshi, PhD  |  Imaging Scientist
Integrated Cellular Imaging (ICI) core, Emory University Health Sciences Research Building (HSRB) EG72, 1760 Haygood Drive, Atlanta, GA 30322


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[Sripad Ram-2]

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Hi Gaurav,
Just to confirm, are you referring to 10x genomics' visium platform?

I don't think you are losing RNA due to laser illumination. It might have
to do with the immunofluorescence assay that sample is being subjected to.
Are you using frozen sections? What is the tissue type? Are you doing an
acetone post-fixation step?  Also are you doing any antigen retrieval or
harsh pre-treatment steps?  All of these can induce mRNA degradation. You
can easily check this by just doing a nuclear (DAPI) or membrane (DiI)
counter-stain, image the sample in IF and then subject the tissue to RNA
extraction.

It's not surprising that there is good mRNA recovery from H&E stained
sections, since the staining process is generally not harsh to degrade RNA.

Hope this helps.

Thanks.

Sripad


On Tue, Dec 15, 2020 at 1:11 AM Sylvie Le Guyader <[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 Gaurav
>
> What do you mean by 'degradation of their RNA'? Less dots? Aggregates? Not
> bright enough to be detected? Anything else?
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Blickagången 16,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Gaurav joshi
> Sent: 15 December 2020 00:10
> To: [hidden email]
> Subject: Imaging for spatial transcriptomics platform
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Ce2ca65aaee174a08414108d8a08581ca%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637435842564665720%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=h%2BPvOoAk6d%2FIWFcj1g5M0o%2BybARg%2BhkURUytvwM1jIw%3D&amp;reserved=0
> Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Ce2ca65aaee174a08414108d8a08581ca%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637435842564665720%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=zqOFXR2mRwjj3rkefRCfgPc4K5UaBh2WfV9%2BKwQWwoY%3D&amp;reserved=0
> and include the link in your posting.
> *****
>
> Hi everyone,
>
> Our facility is seeing an increase in the number of users bringing in
> slides with tissues to image for 10X spatial transcriptomics platform.
>
> We have been doing a tile and stitch for both H&E and IF based platforms.
> We did not have any user issues with H&E based platform.
> For the IF based platform, the user sees a degradation in their RNA.
> We are using a Nikon crest spinning disk confocal for imaging, as it does
> a great job at tile and stitch and gives speed. The user is concerned that
> the laser might be degrading the RNA. The time that the tissue stays
> outside during imaging is not of concern as the users were able to get a
> good yield post H&E imaging during which the tissue stayed outside for 90
> minutes. With IF it's staying outside for shorter than that. I do not have
> a reason to suspect that the laser is degrading the RNA as there are a lot
> of variables with tissue processing during IF based spatial transcriptomics.
>
> In general, I want to know your experience with imaging samples for
> spatial transcriptomics and any issues that you may have encountered so
> that we can serve our users better.
>
> Thank you,
> Gaurav Joshi, PhD  |  Imaging Scientist
> Integrated Cellular Imaging (ICI) core, Emory University Health Sciences
> Research Building (HSRB) EG72, 1760 Haygood Drive, Atlanta, GA 30322
>
>
> När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI
> kommer att behandla dina personuppgifter. Här finns information om hur KI
> behandlar personuppgifter<
> https://ki.se/medarbetare/integritetsskyddspolicy>.
>
>
> Sending email to Karolinska Institutet (KI) will result in KI processing
> your personal data. You can read more about KI’s processing of personal
> data here<https://ki.se/en/staff/data-protection-policy>.
>

[Ann-W]

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Hi Joshi

We made our own platform for imaging Spatial Transcriptomics, although I would like to see it improved a little. The platform comprises a back illuminated sCMOS camera, we find the large chip size really minimises the number of images which need acquiring and consequently light dose. We just use a standard upright epifluroescent microscope, since for Spatial Transcriptomics its only necessary to find the fluorescent dots which comprise the array confocal we feel is overkill. The system at the moment has a metal Halide lamp for epifluorescent illumination. I'd prefer to use an LED - but we have those deployed elsewhere in the facility. It does have a decent precision x,y,z stage from Prior which is important.

We use a Quad dichroic and excitation and emission filters, in Micromanager we can adjust the way data are acquired to image H and E image and the fluorescent image on the same camera to save time stitching and registering the files.

We haven't ever had any issues with signal degradation - our challenges are more around tiling (which we fixed with the sCMOS) and registration which we fixed by having a dedicated sample holder for ST, only being used by the person carrying out the work and by screwing said sample holder onto the stage to minimise the drift. I would say that our platform is not dedicated to ST solely we don’t have enough people doing that sort of work. I think if users increased I might think about seeing what our slide scanner can do.

Hope this helps, please feel free to email me if more information would be helpful.

Ann

Dr Ann Wheeler, Head of AIR.
Advanced Imaging Resource, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU
Working Pattern. Mon 9am - 2pm, Tues - Thurs 9am - 5pm.
E: [hidden email]
T: 0131 651 8665
W: http://www.ed.ac.uk/igmm-imaging


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Gaurav joshi
Sent: 14 December 2020 23:10
To: [hidden email]
Subject: Imaging for spatial transcriptomics platform

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*****

Hi everyone,

Our facility is seeing an increase in the number of users bringing in slides with tissues to image for 10X spatial transcriptomics platform.

We have been doing a tile and stitch for both H&E and IF based platforms. We did not have any user issues with H&E based platform.
For the IF based platform, the user sees a degradation in their RNA.
We are using a Nikon crest spinning disk confocal for imaging, as it does a great job at tile and stitch and gives speed. The user is concerned that the laser might be degrading the RNA. The time that the tissue stays outside during imaging is not of concern as the users were able to get a good yield post H&E imaging during which the tissue stayed outside for 90 minutes. With IF it's staying outside for shorter than that. I do not have a reason to suspect that the laser is degrading the RNA as there are a lot of variables with tissue processing during IF based spatial transcriptomics.

In general, I want to know your experience with imaging samples for spatial transcriptomics and any issues that you may have encountered so that we can serve our users better.

Thank you,
Gaurav Joshi, PhD  |  Imaging Scientist
Integrated Cellular Imaging (ICI) core, Emory University Health Sciences Research Building (HSRB) EG72, 1760 Haygood Drive, Atlanta, GA 30322
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