Sv: Imaging for spatial transcriptomics platform ***COMMERCIAL RESPONSE***

[Garner Oliver, BergmanLabora AB]

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Hi all,

***Please note that we are the distributor of Nikon microscopes here in Sweden***

Having worked with several manufacturers and research groups working with this here in Sweden I thought it might be of interest for us to post some information.

The first configuration (>2 years ago) was Nikon Ti2 (18mm FOV) + Prior H138 with 8-slide holder + sCMOS (Zyla, Flash4, etc.) + Spectra-X
The current configuration is Nikon Ti2 (25mm FOV) + Standard motorised Nikon Ti2 stage + BI-CMOS (Sona 4.2-11 or Prime 95B 25mm) + Spectra III

In order to speed things up, the optimal configuration utilises the 25mm FOV of the Ti2 on one of the large sensor BI-CMOS cameras. One of the groups even uses a 30mm ROI on the Sona 4.2-11 as they are happy enough with the homogeneity (using CFI60 Plan Apo Lambda 20x or 40x dry lenses). We've tested several light-sources and settled on the Lumencor solutions as they have high output and specificity. We use the Semrock filters designed for the Spectra III:

https://www.semrock.com/SetDetails.aspx?id=3708
https://www.semrock.com/SetDetails.aspx?id=3655

I would prefer a different CFP/YFP/mCherry set better designed to hit the gaps in the DAPI/FITC/TRITC/CY5/CY7 set, but this is what we have at the moment.

Please note that we have a specially designed LED-only Spectra III here in the EU. The standard Spectra III has lasers in for the CY5 and CY7 lines. You can also hardware trigger (using NIDAQ) the Spectra III which will shorten the acquisition times.

We also have a couple of systems running Hamamatsu Fusion BT for higher spatial resolution (6.5µm pixels) with emission wheels for better specificity. Here, we get 22mm FOV which is also pretty good for this configuration.

Obviously, the hardware autofocus really helps with the acquisition as does using the functions found in the JOBS module of NIS-Elements.

The datasets will be large and the latest PC (HP Z4) we use has 4x 2TB SATA SSDs in RAID0 (1JQ17AV). We did come up against some issues with storage capacity on the Z4, so I'd recommend sticking to the components and configurations listed in the HP Z4 Quick Specs guide. Also, I'd recommend a Quadro RTX4000 in the acquisition workstation, but if you're going to do some NIS.ai training go with at least an RTX5000.

The above recommendations are based on our experience with NIS-Elements, using different acquisition software will have an impact on the recommended PC spec's (Intel + Nvidia vs. AMD Ryzen, etc.).

I hope this helps!

Vänlig hälsning / Kind regards
Oliver Garner
Försäljningschef / Sales Manager, BergmanLabora AB

Direct: [phone]
E-mail: [hidden email]
www.bergmanlabora.se



Part of Addlife AB (publ)  



-----Ursprungligt meddelande-----
Från: Confocal Microscopy List <[hidden email]> För WHEELER Ann
Skickat: den 16 december 2020 14:00
Till: [hidden email]
Ämne: Re: Imaging for spatial transcriptomics platform

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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


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From: Confocal Microscopy List <[hidden email]> On Behalf Of Gaurav joshi
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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 The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.