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Greetings, Thanks for being here.
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My name is Mikhail Okun and I'm a field application scientist from ThermoFisher and I will present our EVOS S1000 spatial imaging platform today and tell you how it can help you to make your everyday spatial biology tasks easier.
0:30
Well, I guess by now we are ready in the afternoon.
0:35
There is no big need to explain how important spatial biology can be because I guess you have heard already a lot of praises to the methods, how it can show us some correlations between some cellular patterns, how it can help in developing new medicine and so on.
0:55
It has been named the method of the year by nature methods in 24, right?
1:00
So obviously a lot of importance is present there.
1:06
And that's exactly why ThermoFisher is stepping in into this area.
1:14
So when it comes to fluorescence microscopy and analysing multiple markers in the sample, there is a variety of methods that is available.
1:23
So the conventional, what we call it microscopy is agreed to because it's fast, it's easy to perform.
1:30
But the problem is that usually we are limited by the number of markers, which means that not so many cell types can be identified using these methods.
1:40
Then there is the cyclic imaging and labelling which has the benefit of having a really high number of targets.
1:48
However, sometimes it's lacking the throughput because of the longer time and there could be some issues with tissue damaging depending of course on the way how the tissue is handled.
2:01
And in some cases because of the multiple rounds of imaging, it could require some post-acquisition processing.
2:09
So the other option is what we call the spectral imaging, essentially analysing and obtaining all the labels in one imaging run.
2:21
The benefit here is that it's faster and it preserves the tissue better.
2:26
The harder the more difficult moment here is that we need to do unmixing in many cases because having multiple dyes would mean that they start to interfere and we have a lot of bleed-through.
2:40
This is what can be solved successfully by using unmixing.
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The other problem is that it could be a more difficult assay setup with so many markers.
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Again, there are tools to actually deal with that.
2:55
This is what I will show in my slides.
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So as I said, unmixing is the solution to go when you have overlapping dyes.
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In this example that you can see on the slides there are three dyes that are really close together.
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So we have the 488, 514 and 555 dyes and without unmixing, you would see some structures in your sample which would be visible across all the channels, even though we know that they should only be positive for one marker.
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But unmixing is a robust approach which allows us to get back to these original, the real location of the markers in our sample.
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And this is exactly what we will be doing with the EVOS S1000 system.
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This is how it looks.
3:45
I mean, nowadays most microscopes aren't really very fancy anymore.
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They mostly look like yeah, cubes or rectangles.
3:54
We have not an exception here.
3:56
And as I said, the EVOS is a brand with history already.
4:01
And throughout the decades of developing different EVOS models, we always followed one simple rule.
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It was all about being simple and easy to use.
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We wanted fluorescent microscopy to be accessible to a wide range of scientists, not only core facility managers or that one person that was trained in the lab and that is allowed to approach the microscope.
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EVOS was always about, you know, being simple, easy.
4:30
And this is the values that we also apply here when working in the field of spatial imaging.
4:38
So what are the main challenges that we face when working in this field?
4:44
So first of all, as I said, the unmixing that is in many cases limited to certain dyes and then you are kind of bound to be using these dyes, which is not always a good thing.
4:56
The unmixing itself, once you set up your assay, the unmixing is in many cases a black box approach, which means that you have your images and then something happens and then you get your result without really understanding what happened.
5:10
What is the quality and the value of this unmixing then?
5:16
Yeah, well-being intuitive is of course important.
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We want people to understand and easily learn how to use the instrument and the software.
5:25
And then when increasing the number of targets in your assay, starting from a certain point, it gets rather complex in many cases.
5:37
We wanted to go away from this complexity into a really straightforward, make it simple workflow essentially.
5:45
So the limitation in terms of the dyes in the S1000 instrument, you can use the Alexa Fluor dyes, which I guess probably everyone knows, right?
5:58
We can use the Alexa Fluor Plus.
6:01
Plus we have the other wars which I will talk about later, which are our TSA amplification reagents and also Opal dyes which are known in that field, right?
6:11
We have protocols which are validated for all of these dyes.
6:16
Dyes can be combined as well, as long as they don't interfere in terms of the Spectra.
6:21
Of course, we can use primary conjugates and the amplification reagents both, and we can also combine them within one assay.
6:30
So really, a lot of flexibility is there for you to choose from, right?
6:38
So how do you choose what you want to use? In any assay there is benefits and difficulties of course.
6:46
So the primary conjugates are really very fast in terms of sustaining, but they are maybe less flexible because, well, you just have less choice here.
7:01
Then in terms of the target abundance, of course, TSA simplification reagents, they would work better with lower abundant targets.
7:11
Well, simply because the amplified targets of course.
7:14
On the other hand, the primary conjugates are not as good in amplifying weaker targets.
7:21
But again, the speed is the number one benefit of course there.
7:25
And as I said, you can combine.
7:26
So if you have some low bound targets, this can be used with the amplification reagents, whereas others can be labelled with primary conjugates.
7:37
This would already, you know, contribute to a faster workflow in general.
7:46
So the black box of unmixing is a thing that we have heard from many customers and we wanted to go away from this.
7:56
How unmixing works in 2 words?
7:58
Well, it's kind of tricky to explain in 2 words of course, but the idea is that the user is supposed to create a single colour controls which are samples of the same tissue type, but only stained with one dye at a time, including one that which is completely unstained.
8:14
This is where we measure the outer fluorescence then, and you are going through a series of calibration steps.
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When the samples are ready.
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They take like 15 minutes maybe and then an unmixing matrix is created by the instrument, by the software.
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But in contrast to many other platforms, we give you an unmixing report, a comprehensive document where you can work through a lot of data and statistics.
8:45
For example, what you see here, the raw versus the unmixed images from your sample, really looking what we see seeing is believing how the bleed-through adjustment channels disappears as you apply the unmixing onto your samples.
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This is just a part of the report.
9:08
There's many more data like the overall signal intensity from the unmixed images and so on which gives you really an idea of how well your mixing went and if there is anything wrong you will probably see it from the data.
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And there's a troubleshooting section which helps you to investigate and if needed, redo the calibration to really get the unmixing quality that you expect to get from your samples.
9:40
In our case, it's totally irrelevant how many dyes you use for the unmixing, whether it's 6 or 9, which is the maximum that we support in one round with the S1000 system.
9:53
It's really very straightforward process.
9:57
The unmixing per se is happening on the fly, so as you click the big blue capture button in the interface, it will simply unmix immediately at the same moment.
10:07
And then of course, you can tell the software to save the raw data as well as the unmixed data for you to choose later which ones you want to keep for your analysis, right?
10:18
It's up to you.
10:19
It's all part of the acquisition.
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There's no post processing involved at this step.
10:23
So really very simple to set it up.
10:26
The training literally takes like less than a day and you are ready to start working with the system.
10:36
Good.
10:36
So this brings me also to the point of being intuitive in general.
10:40
As I said, the EVOS has all been about simplicity, ease of use, and we keep the same values here.
10:47
The interface of the software, I mean you can see it here.
10:50
We have the slides on the left side, an overview of each slide on the right side of the screen.
10:57
This is where you also draw your areas of interest, which will be scanned later.
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In the middle, you're having either the life image which you see right now when the lights are on, or the resulting scan from your area scan, objectives, focus channels, all, they're really one after the other.
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Very simple, very easy to learn.
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No need to be a professor or whatever to be able to run the instrument.
11:22
Very straightforward.
11:26
A couple of words about the reagents that we offer with the system.
11:30
So the Aluora is our spatial amplification solution, essentially reagents that would amplify your signal based on tyramide signal amplification.
11:44
So you have a primary levelling antibody and then an HRP labelled secondary goes on top, which is then used in an amplification step, which creates a covalent bond of fluorophores like a small fluorophore explosion around the area of the primary antibody attachment.
12:03
And this is a covalent bond.
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So it stays there forever.
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It can't go away.
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It's really very robust.
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And this is of course repeated then for every single primary.
12:13
It can be a long process.
12:15
So roughly two to three days in the manual workflow for nine-plex an automation would allow it to do that overnight approximately.
12:25
Then of course you have excellent signal strength because this is where your signal is actually amplified.
12:31
The drawbacks as I said is maybe the time and in some cases you might want to remove the digital label.
12:38
Well, this is not possible here because of the covalent bond of course.
12:45
But then of course, the images look incredible because of the signal amplification.
12:50
Here we have some samples of some ductal carcinoma.
12:56
Each of them has a nine-plex label and you can see the resolution.
13:01
So this was taken with a 20x, a 0.8 NIR objective on the S1000 is really great.
13:09
This can be, this can go straight into image analysis from that point, the other choice would be the primary antibody conjugate.
13:22
So essentially the primaries which are connected with the dye.
13:28
In this case, we go down from 2 days to one hour of labelling.
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You simply apply the cocktail of antibodies to a sample and you're good to go right.
13:40
Much faster, of course, efficient, robust, but of course there is only a limited menu in the catalogue for the ready to go primaries and there's no signal amplification.
13:54
However, we offer kits where you can perform your own conjugation.
14:00
So you can take your primaries, you can take the fluorophores and conjugate them yourself, which gives you your flexibility.
14:07
And we also offer a custom on demand conjugation services if you wish for those to be performed.
14:16
And this of course is great as well here for cancer samples.
14:23
So we have here some adenocarcinoma samples.
14:27
You can see the healthy on the left and the cancer on the right with all of the cancer specific cells which become active in the image on the right.
14:39
And you could also go closer in on the lymphoid structures.
14:44
And then so in this case the analysis of the images was performed with Halo.
14:48
You can quickly get the composition of the tumour cells in such a sample.
14:54
Yeah, this is what can be done with the S1000 and the primary conjugates.
15:04
All right.
15:06
Then again, as I said, and we can combine the Aluora and the primary conjugate.
15:11
So this is an example here of the two staining methods combined for greater versatility.
15:18
This is also possible.
15:23
And we can also combine RNA targets and protein targets, no problem at all.
15:32
In this case, you see here we have a couple of our targets together with some other protein targets.
15:39
It's nicely visible.
15:40
Again, this is ready to be analysed with any sort of image analysis software.
15:50
So what are the main takeaway messages here?
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The benefits of the S1000 system, we are not fixed to a certain dye set or type.
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We're flexible for different tissues, different labelling methods.
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It's really simple.
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The whole process of actually creating the unmixing matrix is really fast and reliable and also more or less transparent because you do get a report with all the dos and don’ts and the position is really fast.
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So we have the hardware in the instrument which allows us to be really quick.
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One square centimetre of your tissue with a 20x objective with a 9-plex would take about an hour.
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And then this is the tool that you would want to use for making simple spatial biology, right?
16:48
Feel free to meet us at booth 63 where we can show you the simulator of the software in action.
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I can show you how simple it is to perform scans, to view the protocols and so on.
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And if you need more information, then you're welcome to visit our website.
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There's AQR code and also the link to learn more about the system, some resources and so on.
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Thank you very much.