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Hi everyone, I'm Lilia.
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Lilia Draganova.
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I'm the Imaging and Flow cytometry instruments health specialist at Miltenyi. Today I will entertain you with a few minutes of spatial biology and our instruments for spatial biology, which is the MACSima platform.
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I know we should have time for burning questions afterwards, but if we run out, please come upstairs to the booth where we have an instrument and we'd be happy to chat with you about this.
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Before I go into too much details on the spatial side of all the spatial instruments, I just want to give a brief overview of the rest of the imaging portfolio.
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Also, what was pointed out in the talk just before me, I mean, I'm a bit biassed.
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I'm a cytometrist in background.
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I think of cells as dots on a dot plot or pretty images on a 2D image.
0:50
But we shouldn't really forget the 3D, I think Burn had a very nice image of the cell turning around.
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And obviously, you know, when you talk spatial, we should be aware about the 3D.
1:00
Hence just quickly to note other imaging instrument, which is the auto microscope Blaze.
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This is a light sheet microscope for 3D imaging.
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On the other side, as we've got the MACSima platform, which is for the 2D deep phenotyping spatial biology with the Blaze with a 3D instrument, this is where we can look at whole organs, organelles, but actually even an entire mouse.
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And on the other side, with a Maxima, we can look into technically unlimited number of markers to deep phenotype our tissue.
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The reason I'm mentioning this is that we actually have a workflow that combines the two.
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So we talk about spatial biology, but actually which section do we pick?
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That's a different challenge that we often underestimate.
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This is where the blaze or any 3D immunology can help or any 3D imaging can help us, right?
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We can pick actually a section that we want and then we can go back to 2D spatial and there then pick the section to narrow down.
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So we've got a workflow combining these two obviously techniques that we'd be happy to chat about with you upstairs in the booth.
2:07
But then going back to the spatial side of stuff.
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And I guess you all know this better than I do, right?
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Many of the applications for spatial biology, why would we want to do this?
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It can range from, you know, phenotyping to neighbourhood analysis, different cell interaction or indeed the functional states of the cells.
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And again, all of these different applications, they fall in broadly into both the research and discovery side of science, going into the translational but even also the clinical side.
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So obviously talking about biomarkers, this is where we've gotten, you know, screen large samples specifically for what are the biomarkers for these different applications.
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So again, you I'm sure you know this as well as anyone else, the challenges that we all have to deal with when working with spatial biology.
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We've got complex instruments.
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They all have their good and bad sides.
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They all have their strengths and weaknesses.
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But often it can be very complicated to start up these experiments.
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We're dealing with different samples and different fixation methods than the reagent side of things.
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Not everything works together perfectly.
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There's many challenges and if we're talking about spatial and many markers, as long as it takes to optimise 1 antibody, then multiply this by however many markers you want.
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So it can be a big challenge.
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Software.
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It's all good that we talk about big data, but what do you do with the big data once you've generated it?
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How do you handle this?
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And then also the scientific support and this is where really we come in and we try and actually help customers.
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We try and support you for the whole workflow.
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Those of you familiar with Miltenyi will know that we're very big and the whole workflow solution, we try and support people from the start to the end of their experiments.
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So how do we address this with the MACSima?
3:49
So with the instrument, as I said, every instrument can be complex and scary initially, but believe me, mildly biased here, but the Maxima is a very simple instrument to use on its own.
4:00
The instrument is marketing.
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Hate me saying this, but it's quite a boring instrument.
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It's a big box, it does its thing in the background, you don't really see much.
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You don't really have to do much, it's just generating the data for you.
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Then on the other hand, we've got these MACSima sample carriers.
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I'll go into details in each of these in a bit, but this is what allows you to use pretty much any sample that you need really.
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We can also combine protein and RNA on the same section and it's non-destructive.
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So you can follow up with what you need afterwards.
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And also we do have a solution for the software.
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So again, a lot of these platforms, a lot of these spatial data generating platforms will generate the data and then you're kind of left alone to figure out this free open source software with different solutions that require several people from the bioinformatics department to try and make sense.
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You can still do this, but we can also help you with a software that will give you analysis from start to finish.
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It will not have every single solution, but it can get you from start to finish.
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And then again, we do have the people that are there to help you throughout the whole process.
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So going back a bit, I suppose, and what is the MACSima?
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It uses this so-called mixed technology.
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It stands for Maxima imaging cyclic staining.
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So it's in the name.
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We're using cyclical immunofluorescent staining, so with standard e-mail fluorescence you can go up to five.
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With some systems, I guess up to 7 or so parameters.
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It becomes a challenge and obviously a limitation to how deep you can phenotype with a maximum, as I said, technically or unlimited with routinely have panels over 200 markers.
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We've done over 400 markers, but please don't do this, it's too much data to handle for anyone.
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But technically, you can go as far as you need to.
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How does it work?
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As we said, it's cyclical staining, imaging and erasing.
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It goes through staining with up to three antibodies.
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Then we image this, and then we erase the signal.
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Importantly, we also take an image after the erasure.
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So this is your QC image.
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This background is subtracted from the subsequent cycle, but this is data that you always have as we think it's important that you have your full QC data.
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And then again, we can do this repetitive repetitively time after time.
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Going into more details on the antibody sides with maxima, you can use obviously here we're starting with staining, we're using amino fluorescent antibodies.
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We tend to use up to three antibodies per cycle.
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It can be 50 P and ABC, but this can be also their variations in colours.
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And we do prefer direct immunofluorescence, but you can't also do primary and or secondary and secondary.
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So we do the staining.
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This can be incubated for whatever time you decide.
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And then we image, we generate OME-TIFF files that can be used on anything.
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And then we erase using two different mechanisms.
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So we can either photo bleach, which is very gentle to your sample, so we're not chemically treating your sample, or we can use this enzymatic Miltenyi specific for some of our agents kind of signal erasure, which will remove the signal across the whole sample.
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And then again, we just go through cycles of this repeatedly into a bit more details on the instruments.
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Again, the instrument is upstairs, so you can see it.
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It's a bench top blue box going into the different bits and pieces.
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The computer, actually the instrument has two computers.
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One is controlling what is happening, the other one is already actually pre processing or analysing your data.
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So it's doing all the stitching, it's doing the background subtraction.
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So when you come back, when the experiment has finished, you actually have data ready to open and analyse.
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The instrument is fully automated.
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Like I said, that's what I mean, it's pretty boring to do.
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There's not really much.
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It is doing everything.
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It's got this liquid handling system that it will prepare to antibodies on your sample, incubate for however long you need and then wash off and do the rest.
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Ultraprecise stage.
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Obviously it's moved.
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That's again, you know, the stage that powers the automation.
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It moves the sample to wherever it needs to be, the liquid handling station or the microscope.
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And then in terms of microscope, we've got 3 objectives.
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So we've got a 2X objective for an overview scan and we've got two different 20X objectives.
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Whether or not you're using a standard 1 millimetre, 1 centimetre glass slide or if you use a 1 millimetre, sorry, slide or effectively a cover slip thickness slide.
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Going back to the sample care, as I mentioned earlier, so you have flexibility with whatever samples you need as long as they're mounted on a glass slide in terms of the tissue sections.
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So we've got these MACSwells and effectively this creates a reaction chamber on your on a slide.
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So you don't have to span antibody on the whole slide if you don't have to, if your tissue is not.
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So you can create a smaller reaction chamber to keep your running costs low.
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We also have an option with 24 well plates for cultured cells.
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And we do have a solution for cell suspension if you need to do this.
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But really at this point, it's like you could also do flow cytometry.
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And again, you're not limited in terms of the FFPE PFA fixation assays only.
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You could handle your sample however you need.
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And this is how these MACSwell work effectively.
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Your glass slide is pressed between the plastic frames, again, like I said, creating this little reaction chamber.
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You can use any standard glass slide or you can use our high-resolution slides, excuse me, which effectively it has cover slip thickness in the middle, but it's surrounded by a normal 1 millimetre slide to make it easier for you to handle and not break it.
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As I've said, you can use any antibody.
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We do prefer the primary conjugates.
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Obviously, we have a large portfolio that we'd love for you to use of our recombinant affinity antibodies, but we can also use this Miltenyi releasable dyes.
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This is where we can use the second method of releasing the signal and we can mix and match between the two.
10:02
But actually it is an open system throughout the instrument and also the software as you'll see in a bit.
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So here you can use any antibody.
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It doesn't have to be Miltenyi and it is just any FITC, PE and APC antibody.
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And this can be liquid antibody so you can make up your own cocktails, or it can be one of our predefined panels.
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So we have these dried antibody plates effectively it'll take your hands on time to 15 minutes on the instrument, probably less.
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You plug and play, you put your sample, you put your antibody plates and that's it.
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You can walk away the instruments going to do the rest.
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With these antibodies, with these predefined plates, this is one example.
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We've got several flavours of it.
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One of it is called the immunology panel.
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We've got 61 markers here looking obviously on immune oncology.
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So this is an example.
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We've got tumour, intratumuor and it's a kind of distance, a peritumour and then distance cells.
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We can identify multiple cell populations and again, we have tested this on a number of different tissues.
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And yeah, this is one of the very popular panels.
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It's very easy to use to get started to really generate immune data.
11:21
As I mentioned earlier, we can also do RNA and protein on the same section.
11:29
So yeah, we can do RNA on the same section before we do the protein.
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This is what we call RNA Sky.
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It's very easy to use effectively.
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It's still the same.
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We either have a pre made panel or we can have a custom panel.
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And again, we've got this end-to-end workflow.
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Thank you.
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We've got this end-to-end workflow where we can also analyse it with the same software.
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Sorry about that, right.
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So how does it work with RNA Sky?
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You have to use our high-resolution slides because we need to have this thinner glass.
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They don't have to do a few steps off the instrument.
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So these are the sample probe, the hybridization, ligation, amplification.
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And afterwards you pop the sample back into the instrument, you put the resin plates and you go away and it come back to nice data what it's doing.
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In the meantime, it is again the cyclical staining, imaging and erasing of your RNA probes and this is what we get in the end.
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Afterwards, after the RNA finishes, you can then do again as many proteins as you need, and this is what we get.
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We get a same section, the exact same section.
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We've got RNA protein and then we can merge the two together.
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Importantly, this is non-destructive technology.
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So actually you can then take up the slide and take it to something else, you know, be in mass spec or in this case we've got an H&E image that we've taken.
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And again, the applications here, you know, we're not looking at thousands of RNA.
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The idea here is that, you know, you can use a few RNAs in order to, you know, cross validate your methods look at some antibodies, some markers that obviously an antibody will not work.
12:58
You know, secretion markers or in note study the RNA protein Co expression or sometimes when there's just no antibody, right.
13:05
So you have to go with an RNA probe and then looking into the software.
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Like I said, we do have a software that kind of provides all the tools for from start to finish.
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It will not have every single algorithm, but it can get you from looking at the images to everything else that you need to get some meaning from your data.
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So yeah, I'll go into these into a bit more details, but yeah, it's software we actively developing.
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It is actually a bit annoying because we keep adding more features than we need to learn about and tell people, but we do actually get feedback from our customers.
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So things that have already been fed back to us, we can now we now have in place directly from our customers.
13:45
So some of the features that we've got as we've got tools to visualise, export the data, identify the cells so we can segment, we've got different ways to characterise and phenotype.
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So we've got different gates, different, you know, dimensionality reductions, clustering, and again, like I said, to export in different ways for publications.
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So let's hope the videos work.
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Now there you go.
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Again, I can show all of you this upstairs if you come to your booth, but effectively this is what the software looks like.
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You can see that we've got different channels, different markers on the left hand side and we can quickly go between different markers either one at a time or display multiple markers at the same time.
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Once we've well, once we've segmented the cells, which I think is actually in the next video, we can then handle this as any cytometry data.
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We can gate.
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We can.
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In this case, we've got a U map.
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We can gate on the U map.
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You can see that we can display different population, so back gate on the same images.
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And then we can back gate both on the dot plots but also on the image.
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And again here just another short video going into a segmentation in a second.
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So again we can display one or multiple parameters at a time.
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And at any point again you can export the images.
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You can export, for example, the raw data in terms of an FCS file, CSV file.
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We've got different QC tools.
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Again, this is the segmentation where you can see you've got nuclear and membrane segmentation.
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And again, afterwards there's many different features that are measured and different tools to analyse the data.
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But again, you've got specific questions.
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Please pop upstairs and we've got the software running a demo data set.
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This is just an example of a gating.
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Again, this back gating and in addition obviously to the instruments and to the software and all the agents that we provides, we do like to highlight this actually we also here to support you in terms of Technical Support.
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The trainings are available either on site or for you to come to our facilities.
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So we have these application streams, but importantly we also have R&D.
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So Miltenyi expense 1/4 of our team effectively is doing R&D themselves.
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So these are tools that we use and that's why we try and improve and make sense that make sure that they work for people that not necessarily have to be a bioinformatician or super experienced user.
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And obviously you can listen to me all day as I said several times with some mild bias involved.
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But actually, we do have several obvious testimonies and people across the world who use these instruments, and we'll be happy to share their stories in more details or we'd love to put you in touch.
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In addition, you can also pop up to Booth 15 where we have the guys from Exeter who also have an instrument and they're happy to tell you about their experience using this.
16:40
So yes, thank you very much for your attention.
