The Attune™ NxT Acoustic Focusing Cytometer incorporates a revolutionary technology, acoustic focusing, to facilitate flow speeds of close to ten times the speed of any other analyser on the market, with no loss in data quality.
So how exactly does acoustic focusing work?
The Attune™ NxT flow cytometer uses ultrasonic waves (over 2 MHz, similar to those used in ultrasound medical imaging), in combination with hydrodynamic forces, to position cells into a single, focused line along the central axis of a capillary. Acoustic focusing is largely independent of the sample input rate, enabling cells to be tightly focused at the point of laser interrogation, regardless of the sample-to-sheath ratio. This, in turn, allows for the collection of more photons for high-precision analysis at superior volumetric sample throughput. The Attune™ NxT has flow rates ranging from 12.5 - 1,000 uL/min. At lower flow rates (12.5 – 25 uL/min), the sample is focused solely via hydrodynamic focusing. However, as the flow rates are increased, the system incorporates greater amounts of acoustic focusing, with it relying almost completely on acoustic focusing at the highest flow rate of 1000 uL/min. What does all of this mean? By pinning cells into the centre of the flow cell, acoustic focusing allows for groundbreaking levels of sensitivity and resolution at high flow rates, not possible on a conventional hydrodynamic focusing instrument (see Figure 1). Acoustic focusing facilitates much lower coefficient of variations (CVs), allowing for greater reproducibility and consistency in data.
To see acoustic focusing in action please click the following link that demonstrates fluorescent particles exiting an Attune™ NxT capillary at high speeds with and without acoustic focusing.
Figure 1. Acoustic focusing versus traditional hydrodynamic focusing as particles pass through a laser. (a) In acoustic focusing, cells remain in tight alignment even at higher sample rates, resulting in less signal variation and improved data quality. (b) In traditional hydrodynamic focusing, increasing the sample rate causes widening of the sample core stream, resulting in increased signal variation and compromised data quality.
Will the Attune™ NxT fit my lab’s needs?
The Attune™ NxT is designed to accommodate an array of experiments. Being a modular unit, the Attune™ NxT can be upgraded (on-site) from a 1-laser, 4-colour system, to a 4-laser, 14-colour power house. As a group develops and moves into more complex multi-colour experiments, the ability to upgrade to as many as 4 lasers, means that they are not limited to the number of lasers opted for upon purchase.
The Attune™ NxT is extremely compact in size (40 x 58 x 43 cm), with all fluids being stored within the unit itself. The internal storage of fluids is made possible due to its nominal use of sheath fluid (~1.8 L/day), a direct result from its use of acoustic technology.
Spending a fortune on counting beads in order to get absolute counts for your data? Samples on the Attune™ NxT are delivered by a positive displacement syringe pump for volumetric analysis meaning that all events are automatically counted and absolute counts can be viewed with the simple click of a button!
The Attune™ NxT was designed for ease-of-use, ideal for any environment where various levels of flow cytometry experience are found. The bandpass filters of the Attune™ NxT are user-exchangeable, easily slotted in and out of the optical bench depending on the user’s needs. The Attune™ NxT software is designed to provide powerful data acquisition and analysis using an intuitive, user-friendly interface. Experiments can easily be set up, customized and saved for future studies. Automated compensation and maintenance features make flow cytometry easier than ever before!
For higher throughput of samples, the optional Attune™ NxT Autosampler is available, which allows for more rapid processing of samples. Mixing by aspiration instead of shaking, the Autosampler ensures homogeneity of the sample and maintains cell viability. The Autosampler is compatible with 96-well, 384-well, and deep-well plates. Its intelligent probe design minimizes clogging and carryover and prevents damage to the instrument. With a single rinse between samples (which is default on the unit), the specified carryover is reduced to as low as < 0.01 %, allowing for revolutionary levels of data consistency. In practice, customers routinely obtain carryover of < 0.001 % with the Autosampler.
Why not have a look at the Attune™ NxT yourself by following a link to an interactive virtual demonstration here
What else sets the Attune™ NxT apart?
The Attune™ NxT is ideally suited for rare event analysis. When looking at rare sub-populations of cells, samples which could take hours to run on a conventional hydrodynamic instrument can be run in a fraction of the time on the Attune™ NxT. Such samples can only be feasibly run on the Attune™ NxT due to its cutting-edge use of acoustic technology to reach such high flow rates with no loss in data quality. The high speeds at which samples can be run opens up the exciting possibility of obtaining data from experiments which were unfeasible up until now.
To quote one Attune™ NxT customer:
“The problem now is not finding the needle in the haystack, but deciding which haystack to look at.” – Prof. Andrea Cossarizza, University of Modena, Italy.
The high sample rates achievable by the Attune™ NxT allow for simplified sample preparation, reduced protocol times, and minimised cell loss. Standard methods for isolating and detecting leukocytes in whole blood are time consuming and involve significant sample manipulation prior to analysis. These steps can lead to sample loss and alterations in cell physiology. Significantly higher sample collection rates allow the Attune™ NxT flow cytometer to deliver a no-wash, no-lyse protocol to minimise cell loss and simplify sample preparation in whole blood immunophenotyping. Higher sample collection rates are also beneficial for samples that are inherently low in concentration and for precious samples that require that entire sample be analysed to collect sufficient events. To read more on no-wash, no-lyse detection of leukocytes in human whole blood on the Attune™ NxT Flow Cytometer please click here.
Another striking feature of the Attune™ NxT is its non-clogging fluidics. Anyone with flow cytometry experience can relate with the frustration of getting a clog, a common problem with any conventional flow cytometer on the market, which can result in the loss of sample (and loss of precious time!). The fluidics design of the Attune™ NxT makes clogs a thing of the past. Thanks to its unique means of focusing samples, the minimum diameter within the fluidic lines of the Attune™ NxT is 200 um, making clogs close to non-existent. Research groups have reported to have routinely run extremely difficult samples with ease (e.g. nematodes, milk, and brain tissue samples).
A further innovation incorporated by the Attune™ NxT is its use of flat-top lasers. Most flow cytometers use lasers that have a Gaussian emission profile, where the intensity increases until it hits a maximum. The maximum window for alignment is very small, so alignment needs to be very precise. Gaussian lasers have very little flexibility and may yield poor results due to slight shifts in the laser alignment, leading to loss of sensitivity and high CVs. The flat-top lasers used by the Attune™ NxT have an intensity profile that allows a much wider window of alignment. With these lasers, slight shifts in the alignment do not affect sensitivity and CVs, because they have a higher tolerance for misalignment, allowing them to maintain high sensitivity and low CVs.
What kind of feedback have you gotten from customer visits with the Attune™ NxT?
Every customer visit I’ve carried out to date has involved a PowerPoint presentation followed by a demo of the unit/running of samples. Upon completion of the PowerPoint presentation on the Attune™ NxT and what it’s capable of, the general consensus is always the same:
“High speeds, low CVs…….that goes against everything I’ve ever learned about flow cytometry! From day one I’ve been told to never use high flow rates unless I want high CVs and poor data. I’ll believe it when I see it”.
Following each presentation I’ve gone on to run samples and demonstrate to people that this is indeed possible. Suddenly the cogs start turning and it dawns on people what this means; the possibility of saving countless hours running dilute samples, the possibility of completely cutting out protocol steps, tighter CVs than ever before, the prospects are endless.
Ease of use and intuitive; the two remarks I have come across most frequently from customers to describe the Attune™ NxT. For someone carrying out flow cytometry for the first time, it can be a very daunting experience. The constant worry of whether you’re suddenly going to get a clog, whether air is going to get trapped in the system, whether the lasers are misaligned, or whether you’ve carried out your compensation correctly. The Attune™ NxT was designed for ease of use. Because of its non-clogging fluidics and positive displacement sample syringe, clogs and worries of air in the system are a thing of the past.
Users take to the software instantly. Being based around Windows and Microsoft Office, end users instantly relate with its intuitive features, whether it be copy and paste shortcuts (Ctrl + c, Ctrl + v) or the undo button, or the ease by which the software takes you step by step through set-up of your experiment. Following set-up of an experiment, the software takes the end user through their compensation samples before auto-compensating the full experiment. Following this the voltages for each detector can no longer be altered meaning that the end user can be confident in the results they obtain upon running their test samples.
I have found one of the most popular features of the Attune™ NxT to be the automated maintenance features. All of the maintenance features, including start-up and shutdown, are fully automated. Similar to set-up of an experiment, the software prompts the user what to do depending on the maintenance feature selected (e.g. for shutdown – it will prompt the user to load 3 mL of 10 % bleach, then raise the SIP – all click and go). Why is this such a popular feature? Apparently because no researcher enjoys sitting around waiting on the flow cytometer to carry out a shutdown before they can power it off! The Attune™ NxT will automatically enter sleep mode once the shutdown script is complete. Shutting down the instrument is hence as a simple as a few clicks before the last person running samples on a Friday evening can clock out for the weekend!
Another feature I routinely have end users comment on is the running costs of the instrument. Customers are pleasantly surprised with how much cheaper the Attune™ NxT is to run in comparison to flow cytometers they’ve used in the past. This is because the Attune™ NxT uses very little sheath fluid (1.8 L/day), a direct result of its use of acoustic technology. The higher the flow rate a sample is run on the Attune™ NxT, the lower the amount of sheath fluid that is used. This is because although the lower flow rates of the Attune™ NxT (12.5 – 25 uL/min) are solely dependent on hydrodynamic focusing of sample, each increase in flow rate above this uses more acoustics (and less sheath fluid), with sample being almost completely focused by acoustics at the highest flow rate (1000 uL/min). A number of labs I’ve been to have also saved considerable money by no longer needing counting beads for absolute counting of cells. The Attune™ NxT is volumetric, giving an absolute count for every sample run on the unit (easily accessed by the click of a button in the software).
Lastly, and most importantly, the feedback on data quality and consistency has been superb. I have seen numerous groups run samples on their traditional flow cytometer and directly compare the data with that obtained from the Attune™ NxT. Without fail I have found they have either gotten similar or better data. One customer visit that stood out in particular for this was with a researcher that was looking at an extremely rare subset of T cells from human blood. I was told that on a good day they are happy to see somewhere between 10 and 50 events on a dot plot for their population. The user ran a sample (which was dilute and took 10 - 15 minutes to run before enough events were recorded) on the conventional flow cytometer they had on site, and then ran the same sample on the Attune™ NxT (which ran the sample in less than two minutes). Blown away by the sensitivity of the Attune™ NxT, they said the data on the Attune™ NxT was the cleanest they’d ever seen and for the first time their population of cells was extremely evident.
To read more on the Attune™ NxT simply click on the link here.
On the market for a new benchtop analyser? Why not get in touch and demo the unit for yourself.