Attune Flow Cytometer Features

Attune Flow Cytometers combine ultrasonic waves like those used in medical imaging with hydrodynamic forces to precisely position cells into a single, focused line in the central axis. Enabling cells to be tightly focused at the point of laser interrogation allows the system to collect more photons, helping to ensure data quality regardless of the sample-to-sheath ratio.

Acoustic focusing is useful for cellular imaging on the Attune CytPix instrument, helping to position the cells precisely so that a sharp image is obtained. As demonstrated in these videos, cells imaged without acoustic focusing were fuzzy and out of position, while cells imaged with acoustic focusing were clear and centered in the field of view.

Acoustic focusing also lets your lab rapidly acquire high-quality data. Users can achieve sample throughput rates of 12.5 µL/min or 1,000 µL/min, up to 10 times faster than traditional hydrodynamic focusing systems and acquisition speeds of 35,000 events per second. This means processing all samples—including low-concentration and precious samples—more quickly and accurately with minimal loss in quality.

Acoustic focusing minimizes variation regardless of the sample rate, so you don't have to make the tradeoff between throughput and sensitivity. This is demonstrated in the cell cycle analysis example below, where it's critical to precisely detect differences in fluorescence intensity between multiple cell populations.

To prevent clogging and allow for volumetric analysis, the Attune Flow Cytometers use a positive displacement syringe pump to control sample volume. The system can perform volumetric cell counts in a known volume (gated or total events) and can easily gate out dead cells to count live cells only in a live/dead analysis. Unlike traditional cytometers that operate at a maximum pressure of 15 PSI, Attune Flow Cytometers control pressure at 75 PSI, reducing the likelihood of cell-cell interactions and resulting in clog resistance.

Attune Flow Cytometers are configurable with up to 4 spatially separated lasers and 16 parameters. Spatial separation provides flexibility for multicolor panel design and streamlines compensation.  The system offers superior speed with acquisition rates of up to 35,000 events per/second with high sensitivity to meet a range of research requirements.

High sensitivity distinguishes between dim signals and background, resulting in less variation and better signal separation. Fluorescent resolution coefficient variation is less than 3% for a single peak, and predicted MESF is ≤80 (FITC), ≤30 (PE), ≤70 (APC). Sensitivity in comparison to competitive systems is described below.

The compact size of the Attune Flow Cytometer also provides the flexibility of using it within a biosafety hood. This helps avoid contamination or infection when working with hazardous or unknown samples.

Attune Flow Cytometers feature a novel optical design that delivers first-class reliability and superior performance over time. The flat-top beam profile of the solid-state lasers minimizes the effects of changes in fluidics or optics, which in turn can lead to instability or alignment issues and instrument downtime.

Laser misalignment is a major concern with users of conventional flow cytometers. The flat-top lasers used in the Attune Flow Cytometers have an intensity profile that allows a wider window of alignment over Gaussian lasers used in traditional systems.  The flat-top lasers also have a higher tolerance for misalignment that allows them to maintain high sensitivity and low CVs.

Detection of rare events requires acquisition of high numbers of cells to attain a reliable measure of accuracy. Attune Flow Cytometers allow dilute samples to be processed quickly at sample input speeds of up to 1 mL/min, significantly faster than conventional cytometers that support maximum sample input rates of 60–100 µL/min. Acoustic focusing thus offers a unique combination of speed and quality, cutting the time to collect rare events significantly over long acquisition times.

Attune Flow Cytometers support both high speed of acquisition and high sensitivity to enable easy detection and phenotypic and functional characterization of rare cells. This step-by-step strategy for detecting rare events in our Flow Cytometry Learning Center will help you think through the best approach for pre-analytical, analytical, and data analysis phases of your research.

The camera captures images of events at a rate up to 6,000 images/second and enables users to visually verify events recorded by the detector. Users can view event images in real-time to confirm sample quality, optimize protocols, and record the morphology of each cell population identified for analysis. This is crucial in cell therapy applications but can be applied to any flow cytometry sample.

Imaging can be added without changing existing flow cytometry protocols or reducing throughput. The Attune CytPix Flow Cytometer can capture clear images, even while maintaining standard flow cytometry acquisition speeds. Imaging also benefits from acoustic focusing, which helps to position the cells so that a sharp, centered image is obtained.

By using the image gallery view, users can scan cell populations rapidly for outliers. In this view, doublets and other aggregates stand out, even in cursory review. With the Attune CytPix Flow Cytometer, users can highlight structural features of large populations in record time. This allows adjustment of gates to include cells of interest while excluding aggregates, unwanted cells, and debris.

Morphological information from images adds to the richness of flow cytometry data over multiplexed staining alone. For example, the figure shows an otherwise conventional apoptosis assay using Annexin V and propidium iodide (PI), with added cell imaging to characterize cells in each population, revealing morphologically distinct features. These insights could not have been gained from flow cytometry data alone.