The Next Generation In Acoustic Cytometry

 

Dr. Gregory Kaduchak:  So, today we'll be talking about next generation in acoustic cytometry.  We'll be introducing the Attune NxT Acoustic Focusing Cytometer.

My name's Gregory Kaduchak.  I am the Director of Engineering for Flow Cytometry Systems in Eugene, Oregon.  And I'll be joined by my colleague, Gayle Buller.  She's Product Manager for the NxT system in Eugene also. 

So, if you look here, what I want to do is look at hydrodynamic focusing and how it works in a traditional flow cytometer.  If you look up the upper left-hand corner, you see that you have a sample going up at 12 microliters per minute through a flow cytometer.

One of the key things here is sample positioning for the sample through the laser beam.  So, what you have is the sample hitting a very focused laser.  And you want the sample to hit the laser at the same points through--at the beam.  And you want that because you want the photon flux to be the same at every particle.

If you go over to higher flow rates, as you see on the right-hand side, you see that the core is much larger.  The particles are in a much, much broader stream.  So, you see different parts of the laser.  Intensities are different, so your photon flux is going to be very different. 

What that leads to is broader CVs.  So, in traditional flow cytometry, most of your flow rates are going to be somewhere in the 12 to 60 to maybe 120 microliters per minute due to this limitation. 

With acoustic focusing, what we're doing is we're changing things around a little bit in how we introduce sample and how we position it at the laser.  So, if you see here in the video, you see particles coming up through a capillary.  These are ones that we build in our manufacturing plant for acoustic focusing widgets and things that we make.

But, what you see is particles coming up through the center of capillary of about 350 microns in diameter.  This is the cross-section, but what we're doing is we're giving a--ultrasonic radiation pressure is giving a momentum boost to the particles.  And that momentum transfer is taking the particles to the central core of the capillary and focusing them there.

That focusing, then, is used in conjunction with hydrodynamic focusing to push the--to center the particles within the laser beam. 

So, here on the left-hand side, this is what we do in our acoustic focusing systems.  On the left-hand side what we do is we put our capillary in the sample input, and we do acoustic focusing before we wrap it in a sheath and then take it up to the interrogation zone.

So, on the left, this is what you're used to in your standard flow cytometer.  This is pure hydrodynamic focusing.  So, we're running a sample input at 12.5 microliters per minute.  We're injecting a sample that is acoustically focused, but it doesn't--it's really not needed at this point, and then we're positioning very finely within the laser.

On the right-hand side what you see is a sample input at 1,000 microliters per minute, about 10X faster than most flow cytometers on the face of the planet.  What we're able to do here is we acoustically focus the sample first, then we wrap a small sheath around it and then run it through the interrogation zone. 

And what you see is that, in this application, we're still able to hit that central core of the laser beam, so our photon flux is steady.  With that, we're able, at these high injection rates, to come up with very narrow CVs for our system. 

And that's what separates us out.  So, if you're thinking about running samples that are dilute, if you want to skip centrifugation steps in your sample, this is the system that you can do it with because you're almost 10X faster than anything else on the planet.   

What this has caused us to do or made us do is kind of design our second offering of acoustic focusing systems to be more modular.  So, we've come up with a common platform base for our system.  Here we're going with a one to four laser system that's based upon a common modular platform.

So, we're going to be offering a one laser system with four colors all the way up to a four laser system with 14 colors.  So, we're going to take the entire product line and make it on that same platform.  We're going to build a one laser system, make it the same as the two, the three, and the four as far as that base platform, and just add lasers and PMTs.          

What does that do for us?  Well, from software, it's one software version.  Manufacturing, we build one single laser system and then we add additional lasers and detectors at receipt of order.  The auto sampler that we have is the one that's already been proven out in the field with the initial Attune launch. 

We have a lot of efficiencies here simple from training and service.  So, for training for customers, if you want to buy a one laser system it's one version of software.  If you then upgrade to a four laser system, it's the same version of software.  If you want to buy a one and a four, it's the same version.   

So, it's very simple on the training side.  But, that helps us too in our efficiencies, because when we have a field service representative come out and take care of your flow cytometer, they only have to know that one platform; same with our field application specialists. 

Service is the same thing.  If you buy the low end one laser system, you go up to the high end four laser system, you're still dealing with the same thing.  The fluidics are the same.  The optics are the same.  Everything is identical.  So, there's an efficiency there, but also a robustness as far as the service is concerned. 

So, what we offer is a modular design.  We're offering one to four lasers, so you have a one to four laser configuration that can detect up to 14 colors.  It facilitates a broad range of reagents that you can use on the device. 

The broad range of reagents, what does that buy you?  Well, first, if you go up to the 14 colors, there's a large menu or a large amount of space and different reagents that you can choose from.  In a lot of the studies that we've done, most flow cytometry is done with just four to six colors, and that's the predominance of most experiments. 

So, people just might say, "Why not just buy a six color system?"  If you buy the six color system, you're limited to the six detection channels on that system, the one to two lasers that are exciting those lines, so your menu is reduced as far as reagents.  The other thing that you have a problem with then is compensation.  Your compensation becomes an issue because all your channels are typically stacked spectrally very close to one another.

Fourteen color systems, four lasers, if you have that ability to go up there, you can greatly reduce compensation by choosing channels that are spaced apart.  So, going to different lasers and putting those channels out at different places, you can greatly reduce compensation.  So, even though you're only using four or six colors, you can make things a lot simpler by going to these high multiplexing systems.

And then finally, fully upgradeable.  So, as you see here, we have the one laser system that is based at 488 nanometers on the blue.  Then our two laser system is any combination of what we offer, which is a 488, a 405, 561, and 640.  Then we go up to a three laser system, and the four laser system up from there.

But, as I was saying, the one laser system comes with everything that you need to do that upgrade.  So, what we've copied is what you see in the automobile industry. 

If you want to--you may not buy the upgraded stereo on day one, but what they do is they put the wires and a lot of the infrastructure in place for that upgrade.  And that's what we've done here.  That one laser system has all the optics and everything in place for that.

So, what we can show up with--if you buy the one laser system this year, you can upgrade that to a four laser system in a year or two years.  What we show up with is the lasers and the PMTs.  Everything else is there for the base platform so we don't have to change anything.

One thing I always say is the one laser system is probably the most fluidically stable one laser system on the planet, because it has to be able to do the four lasers when we come out there for the upgrade. 

The next--this slide talks about probably what I would call one of my soapbox topics.  We decided to go with flat top lasers on this system.  And we've gone with flat top lasers to deal with what you see here in the two images.

So, the image on the left, where you see a Gaussian laser beam, that's a standard Gaussian laser like you'd see in a standard flow cytometer.  It's one over E squared.  It's about 80 microns.  And that's intersecting a 10 micron core of particles.  When you do a numerical model of this, you come up with a CV of about 1.1 percent.

To me as a physicist, as an engineer, this is a very unstable equilibrium position.  Any change in the position of that laser beam, any change in the position of that particle core, your CVs can change drastically.  So, anything from vibration, temperature fluctuations, anything that can change these things can send your findings or your data into quite large variations. 

What we've done is asked the question, why do we do this and how can we make this better?  So, we've gone with the flat top, as you see it on the right. 

So, right-hand side, that is an actual laser beam profile that we have on our systems.  And what you see is that you have margin to the left and to the right of that particle spot or that particle core coming up through there. 

If you misalign the system by 15 microns, as you see for the Gaussian profile, which--that's the perfect Gaussian.  You wouldn't see that in reality.  But, that's an 8 percent CV now.  If you go to the flat top with the 15 micron offset, you're still at sub 2 percent CV.

So, what we've done is we've really put a safety net down there for your experiments and for your data to compensate for any of these fluctuations you may get in your day-to-day experiments. 

So, our standard lasers that we're doing with this are what we're launching with; 488 nanometers going to 50 milliwatts, 405 50 milliwatts, 637 100 milliwatts, and 561 is going to go at 50 milliwatts.  But, we are going to be changing that up. 

We're going to launch at the end of--in actually third quarter this year.  After the launch, we'll go with custom lasers.  So, we'll have custom powers to offer.  We'll offer higher powers than what you see here. 

And then, we'll be coming up with, as you see in the upper right-hand corner, custom lines.  So, we have offering of other different laser lines in here, and we'll be adding custom laser lines to the offerings also.      

The instrument is very small footprint.  This is one thing that we strived for.  We wanted to make this something you can fit in a hood, fit on the bench top.  If you're trying to manage space and make things simple, this is what we've done.  So, we have four lasers, 14 colors in a box that weighs 64 pounds.

So, this could be moved by one person.  I recommend two given the size of the investment in these boxes.  But, it doesn't take three people or a lifting crew to lift this thing.

The other thing is doesn't require is the dedicated circuit.  The power consumption on this is very low, so you can plug it into a standard outlet and get the power that you need.  So, you don't have to dedicate a space or a location for it. 

Specification wise, it's what you've come to expect from a flow cytometer.  Acquisition rate for events, approximately 35,000 events per second is the high end that you'll get up to.  Particle size range, half a micron to 50 microns is the particle size range. 

And you can see up in the upper right-hand corner a plot of that.  That's a--that's one of the standard instrument's outputs that we have, forward scatter versus side scatter.  You can see the .2, the half, and the .8 going up there. 

Fluorescence sensitivity, MESF is 80/30/70 for FITC, PE, and APC.  One thing I will say is that everything that comes off of the factory floor, we'll be testing all of these specifications.  So, we're not cherry picking things or testing one and saying it matches for the lot.  Every instrument being shipped out to a customer will be going through all these tests.

Fluorescence resolution, the standard CEN test will be performed.  And you can see data here at 100 microliters per minute, which really is the high end of most flow cytometers, or the nonstandard or the--I would say the broad CV range for most flow cytometers is what's show.

And then finally, forward and scatter--or forward and side scatter resolution.  As expected, you should get your three populations of blood, your monos, your lymphs, and your granulocytes.  And that is one of our specifications here. 

Sensitivity and precision, what we have up here is just showing the 8-peak beads on our system.  Yellow is an up and coming laser, 561.  So, what we're showing you here is the second channel off yellow. 

On the left what you see is the 8-peak bead set off of a very high end flow instrument going at 12.5 microliters per minute under hydrodynamic focusing.  What you see in the middle is our--is an Attune NxT system going at 12.5 microliters per minute.  And you can see the resolution is very good and the CVs are very good as far as the CV--or as far as the 8-peak beads are concerned. 

Then finally, over to the right what you see is 500 microliters per minute, very fast.  That's faster than any flow cytometer out there.  And you still see the 8-peak bead resolution on that YL2 channel.  You see that across there. 

But, you also see the very tight CVs here.  And this is what this instrument does for you.  So, tight CVs, good resolution, good performance.  Great performance, really, when you think about the flow rates.  Taking that a step further, these sample rates are, as I've been saying, about one mil per minute rates, or about 10X faster than anything out there. 

But, the thing that we didn't do well in our first launch and I do want to hit here is that the 12.5 microliter per minute rate, and even the 25 microliter per minute rate, it's purely a hydrodynamic rate.  Acoustic focusing really doesn't play a part here.

So, if you're interested or you have worries about any of the acoustic focusing as far as, you know, my samples--my particle size may be too small or it's a sample I believe in and I work there, we're giving you the exact same hydrodynamic focusing that you've become accustomed to over the last 30 to 40 years.

So, your 12.5 microliter per minute flow rate here is the same as what you get off of the same--the instruments you have in your lab; same with the 25 that we're offering.  What we do do is we take these higher rates and, as we go higher and higher in sample input, we start adding more and more acoustic focusing here to assist with the hydrodynamic focusing.       

So, by the time you get up to 1,000, you have a lot of acoustic focusing occurring with the hydrodynamic.  You still have both.  The acoustic is assisting the hydrodynamic, but you have a large degree of it. 

So, you're starting at one end with hydrodynamic focusing.  We take you all the way to the other end with the acoustic focusing and then have a middle ground in between.  So, we give you everything you're used to and then we give you everything on top of that. 

From there, you can see the data across the bottom at the different flow rates.  You have 12.5 microliters per minute, 25, 100, 200, 500, and 1,000 microliters per minute.  Those are the flow rates we're going to offer. 

These are Jurkat cells with propidium iodide.  And you can see the CVs, the primary peaks, all right around 3 percent.  Data looks very good across all these flow rates.  And that's what we aim to do, and it's really a testament of the ability of what this instrument can perform. 

My final one is a very interesting technique and method that we've come up with, and is very well suited to this instrument, seeing that we can run samples so fast, we can do so much input and then dilution.  We can do a lot of things with dilution here given these sample input rates.

So, no lyse/no wash samples is definitely of interest for these applications and for this system.  So, on here what we're done is we've taken five microliters of whole blood and we've labeled it with two labels, glycophorin A and CD45. 

If you look at the first line of graphs, the first thing you see is 488 forward scatter and side scatter.  And what you see is a pretty well a large blob of events all over.  And that--those are red cells.

The orientation of red cells, you have a--an asymmetry there.  So, as they rotate, they have different scattering cross sections so they take up a large part of the forward and side scatter regime as far as the bivariate plots are concerned.

With the labels of CD45 and glycophorin A, the CD45 will label your nucleated cells there, and then the glycophorin A will label the reds.  If you gate for CD45 positive/glycophorin A negative, you'll pull out the white cells. 

And then what you'll do is get rid of any red cells that are coincident with a white cell, and that'll give you your scattering populations that you see in the final right-hand side.  So, the scatter patterns that you're used to with your lyse type samples you can get by doing this with the two reagents that are described.

The bottom is a little--is an interesting one because it's a reagentless way of doing the same thing.  So, if you take a 488 forward scatter and 405 side scatter, you see that some of the blood populations start to show up.  And you can see the granulocytes start to pop up above the red cells in this plot. 

And the reason for that has to do with the hemoglobin absorption of the red cells.  So, the large hemoglobin absorption at 405 pushes that side scatter way down.  And the granulocytes, which are unaffected by the difference in the wavelength, they stay in their normal spot. 

That allows us, at the 405 and 488 side scatter, to have this offset of the red cells.  So, what you see is the blob down to the bottom--at the bottom right which corresponds to the red cells, and that's due to a differential absorption of the hemoglobin of the red cells.  But, then they now separate from the white cells, which--I like to call that that Q-Tip structure that you see.

So, if you start from the bottom left and start working your way up at a 45 degree angle, you can see the lymphocytes, the monocytes, and the granulocytes.  Gate that out, and then you'll end up with the plot on the right, which is something that you're very accustomed to seeing as far as your blood scatter is concerned. 

So, now you have a reagentless way of doing this.  And what we're offering is--on this instrument is we're going to allow you to purchase a violet side scatter kit in order to get results like this.  We also have other methods to come across this. 

As I said, we're very, very good at this, or very well suited for this simply because of the sample input rates we can run.  We can run very dilute samples, but we can run samples very fast also.  So, when you start thinking about whole blood, you need the real--you need to really get numbers through to get them going.  And we're really fit for that, so this is one application that we're really for. 

We'll be publishing results on this shortly and be giving out several other ways of how to do the reagentless way.  So, you can do this reagentless, non-reagentless.  There's ways to do it with one and two reagents.  And we'll be getting information on that shortly.

With that, I'd actually like to pass this on to Gayle, my colleague.  And she'll be talking more with the--more about the applications and assay space related to this instrument.  Thank you.

Ms. Gayle Buller:  Thanks, Greg, for the introduction.  And it's my pleasure to talk to you about the applications to be utilized on the Attune NxT Acoustic Focusing Cytometer. 

One of the things that we found with instrument development is we really wanted to understand what our customers utilize for reagents and how they're utilizing and using them in their particular applications.  And so, we did a lot of customer interviews to understand what channels people use for what specific reagents. 

So, this instrument is really designed to expand the access to the reagents that are available for flow cytometry.  The most commonly used reagents maybe change depending upon what laser configurations you have on your instrument. 

And what we found is, when people tended to have a yellow laser present on their instrument, they needed or wanted to change the number of channels that were available off of the blue and the yellow lasers. 

So, here in this slide you can see when you have a instrument that says--has, for instance, a blue, red, and a violet laser, the customer has access to four different reagents off of the blue laser, FITC, PE, PerCP/Cy5.5, and PE-Cy7, which gives you four channels.

However, when the customer adds a yellow laser, we found that customers wanted to transfer their PE and PE tandems from the blue laser to the yellow laser.  So, we have reduced the number of channels on the blue laser and added an additional channel onto the yellow laser so they could get optimal excitation of those particular reagents. 

And you can see on the far right panel, this is just a 10 color immunophenotyping using human PBMCs on the Attune NxT looking specifically at multiple T-cell subsets and myeloid cells.  And you can see where you can identify both monocytes using CD14 APC/Cy7 and CD33 FITC and also look at the specific T-cell subsets of both CD4 and CD8 mutually exclusive populations, and then also being able to identify dendritic cells.    

So, one of the big avenues of the Attune NxT is really to be able to utilize many different reagent platforms and really be able to broaden your panel choices.  As Greg highlighted, with a two laser instrument it's extremely hard to develop a six and eight color panel because compensation is really involved here.  And so, by being able to have more lasers on your system, you can really broaden your reagent selection choice. 

Here we show a 10 color stain using mouse splenocytes.  And what our researchers did at the Molecular Probes campus is stain cells with B220 Pacific orange, CD45.2 APC/Cy7, and well--as well as CD3, PerCP/Cy5.5, along with CDA4 Pacific green and CD8 Alexa Fluor 700, and then also included MHC class II Pacific blue and CD11c PE-Cy7.

And we were able to identify, gating on the splenocyte population, the T-cell subsets using the CD3 positive cells and looking at the CD8 positive cells, T-cells, and the CD4 positive T-cells.  And then we're able to take either both the CD4 positive T-cells and then look at those specific regulatory T-cells using CD25 and Foxp3 positive cells.

But, if you looked at the CD3 negative subset, then you could identify a sub-portion of dendritic cells which then could also be sub-classified using CD8 and 11B.  So, using the Attune NxT system, you can easily develop a 10 color panel and identify multiple subsets of cells in a single sample.

So, one of the avenues of having a multi-laser system is that you're now able to utilize your lasers to minimize the amount of compensation you have.  And so, here in this example we're just showing you an example of doing a S-phase quantitation using a Molecular Probes product, Click-IT Plus Cell Proliferation Kit.

And what we wanted to do is look and see if we could have a cell proliferation assay using reagents off of different laser lines to really provide you a no compensation assay.  And here we're utilizing Click IT Plus in the 488 or off of the blue laser, and then did a phenotype using a PE off the yellow laser and then looked at cell cycle using FX cycle violet off of the violet laser. 

And if you might be wondering what Click-IT Plus is, it's basically a BRDu replacement which simplifies the workflow.  So, basically instead of feeding your cells BRDu, you are going to feed them a EDU, a thymidine analogue.  And where this gives you advantage is that you do not have to do any of the DNA denaturation in order to get the click reaction to label the double stranded DNA helix.

So, you really have a quick, fast way to do a S-phase quantitation.  And in the lower right-hand plot you can see that the Click-IT Plus EDU gives you a typical horseshoe shape that you would see with a BRDU assay.  And you're able to quantify the number of cells that are actively synthesizing their DNA content in a no compensation-less fashion. 

But, you might ask what is the size range that you can detect cells or particles on the Attune NxT.  And as Greg said, in our specifications we detect particles from .5 micron up to 50 micron.  So, I wanted to show you an example of cells on the low end of our scale.

And this is a simple no compensation assay for bacterial detection.  We stained E. coli strain D5 alpha cells that were stained with a LIVE/DEAD BacLight Bacterial Viability Kit to determine positive and negative, or live and dead cells. 

And here you can see that we gated on the bacterial population and then did some aggregate discrimination, and then was--were easily able to identify live cells from dead cells and be able to count those. 

One of the nice advantages of the Attune NxT as well is that--the fluidics.  We can do volumetric counting and provide you a concentration measurement.  So, either that live or that dead population, you can get concentration statistics per microliter to figure out how many cells you have that are live or dead.

But, being able to expand into additional laser lines like the yellow laser, which is fairly new and up and coming, or going into custom laser as well, is you expand the ability to efficiently excite fluorescent proteins.  And we know that the yellow laser is able to efficiently excite many of the common red fluorescent proteins.

And so, we wanted to be able to show you some examples of detecting of fluorescent proteins.  So, on the left-hand side of the plot, we're looking at live cell detection of both GFP and RFP using a cell line, a U-2 OS cell, that has been transduced with a product called CellularLight [sic] products for both nucleus GFP and plasma membrane RFP.

And because the cell has been transduced with both the GFP and RFP, on the plot you can see a co-positive population of cells that are both expressing GFP and RFP. 

But, one of the advantages also of using a yellow laser is that it efficiently excites many of the mCherry fluorescent proteins.  And here on the right-hand plot you can see the same U-2 OS cells that were stimulated and transfected with the adenovirus with mCherry.

And you can see those cells that are not transfected with mCherry show up in the negative population, and cells that are transduced show up in the positive population.  So, this is way that you can add fluorescent proteins into your common reagent panels just by adding the additional yellow laser.

Traditionally by flow cytometry we have looked at many of the reagents that are available both in the Molecular Probes catalog and available from other sources.  And really, we were utilizing dyes because we needed them for a particular application, but their excitation and emission profiles may not have been the most optimal for use with some of the common laser lines.

And in this slide, we show you tetramethylrhodamine ester.  And in the upper left-hand corner you can see the excitation and emission profile of TMRE.  And what you can see it is that, if you look at the excitation profile, it looks like, if I was going to excite this with a 488 laser, that it wouldn't be very effective or efficient. 

However, for years we have used this dye for detection of mitochondrial membrane potential because it was maybe one of the only avenues that we had to detect that particular characteristic of a cell.

However, now that we have additional laser lines available, what we wanted to do is look at some of these dyes that may have not been optimal at traditional laser lines and see how they--their performance was affected if we used the appropriate excitation. 

So, scientists at Molecular Probes looked at TMRE both with--excitating with a blue laser, or a 488 laser, and both the yellow laser.  And here you can see in the upper right-hand corner a graph that shows the difference in signal between a treated or a not treated cell. 

And the treated cell was treated with a deproteinator to reduce the mitochondrial membrane potential, CCCP.  And you can see by using the correct or optimal excitation source that you get a much better or higher signal difference using a yellow laser than a blue laser.  So, adding these additional laser lines to your instrument will actually provide you a much broader range of reagents that you can use on your instrument.

But, going into the acoustics, besides your reagents we really wanted to see where our scientists can push the limits of our instrument.  And I actually challenge you to utilize the acoustics platform to see things that you may not have been able to see before. 

Traditional flow cytometers really had limitations around the fluidics and the cell concentrations that can be run on the system.  And the Attune NxT really opens that up for you to use a vast differing array of cell types and cell concentrations to do things that you may not have been able to do on a traditional cytometer. 

On the top you see a example of a rare event experiment where we're really looking at CD34 positive cells in human peripheral blood.  And human CD34 cells tend to be in a very rare population, between .01 percent and .1 percent. 

And so, in order to detect these rare events, you typically have to collect a lot of sample or a large number of samples to find a statistically significant number of cells to really identify this rare event.

And so, the Attune NxT obviously is specifically designed or beneficial to this type of assay because we can run much faster than any other flow cytometer out there.  And so, rare event samples can be run in much less time than standard instruments. 

And on the bottom you can see a quote from one of our researchers, David Cousins in the UK.  And he just indicated that, you know, he's been looking at rare event stuff for a really long time, and with--the Attune has really enabled those type of assays.  So, I really challenge researchers to go out and do things with the acoustics that they really couldn't do on other flow cytometers.

And finally, you might be wondering about how do we drive this wonderful instrument.  And we have also done a lot of research and asked our customers and looked at our customers and how they're utilizing their software programs to really try to make the software as intuitive as possible.  But, we wanted to have something--if a customer walked up to our instrument without seeing it before, have something very familiar to the customer. 

And so, I interviewed one customer.  And when I asked him what kind of user interface he would like to see on an instrument, he said, well, you know, when I program my TV and I need to adjust the brightness, I go to the menu, I pick brightness and there is a slider bar there that I can set.  And then, when I'm done it goes away and I don't see it again.

And so, what we wanted to really do with the software is make it user friendly and provide guidance when you need it and be able to turn it off when you don't, and also have it somewhat smart in the fact that, when you're doing compensation, be able to do things or see the menus related specifically to compensation, but not with a lot of extraneous things that might confuse users.

So, you'll find that the Attune NxT software, there will be something familiar about it.  We use an experiment explorer tree.  So, if you're used to that kind of menu classification for your samples or your specimens, you'll find familiarity with that.

If you've used Word or Excel at all, you'll see that the Attune NxT system is--software is set up around ribbons and tabs very similar to Word and Excel.  You can use standard Word shortcuts on our software as well to copy and paste plots.

But, you'll also see that the instrument itself has a lot of acquisition and analysis features.  We can do standard gates.  The maximum number of events in a single file, we can collect up to 20 million events in a single file.  And then, if you need to go any larger than 20 million events, you can append data onto a single file to get into that, you know, 40, 80, 100 million to really be able to detect that a rare event.

Also, our instrument comes with fully automated maintenance features and automated performance tracking.  So, the time that you spend on your instrument is very little from a day-to-day actual user intervention.  And then, if you operate a core facility, each user has its individual user account so you can track user time logging as well.

And the software itself is fully customizable to the user.  So, the user can define their own default criteria so that they don't have to make a lot of selections in a single day.  They can set it up and have it ready to go when they want to use it. 

So, I hope just by this, you know, screenshot and things you can see that the Attune NxT software is familiar to you even if you haven't seen it, and that it's very intuitive and easy to use.       

Finally, with the Attune NxT we also have a high throughput option here with the Attune Autosampler.  And the Attune Autosampler is a device that sits right next to the instrument.  It simply plugs into the instrument with two fluidics cables.  And that gives you access to both tubes and plates at the same time.

It's this--the autosampler is really designed for minimal clogging.  It mixes wells with aspiration.  And we do it that way to really maintain the viability of your cells.  And the instrument itself and the autosampler is compatible with 96 and 384 standard and deep well plates.  So, you have a lot of selection.  You don't have to buy a plate specific to--from Life Technologies.  You can use a broad range of plates that have been validated for the instrument. 

The nice thing is, because the autosampler sits right next to the instrument, you can run tubes and plates without having to de-install your high throughput device.  And also, the autosampler itself has a--automated cleaning and maintenance procedures very similar to the Attune NxT.  So, the maintenance of the accessory device is very little as well. 

And finally, I just want to direct you to the flow cytometry resources page on Lifetechnologies.com.  We have a lot of resources there from webinars that have--are available on a on-demand service. 

There is a Fluorescence SpectraViewer there.  And if you're doing any compensation at all, that's a great tool for you to have.  Any posters or application notes that we develop for the Attune or the Attune NxT can be viewed and downloaded from this resource page. 

We have some flow cytometry tutorials.  So, if you're doing a lot of teaching on basic flow cytometry, there are some great tutorials there for both fluorescent and flow--fluorescence technology and flow cytometry. 

And then also, we are actively active on Facebook.  So, you can join the flow cytometry group on Facebook and share things with your colleagues, as well as, if you're interested in being able to select different reagents for the Attune NxT, we have a flow cytometry mobile app for both iPhones and Androids and also iPads that you can download.  And it provides you at your fingertips reagents and reagent protocols.  So, as you can see here, this is a great resource for you, especially if you're in a teaching environment.

And I thank you for your attention.