Intelligent image-activated sorting of large cells enabled by elasto-inertial focusing

Image-activated cell sorting (IACS) enables high-speed sorting of cells based on real-time analysis of their images, providing a powerful means to link cellular morphology and function at the single-cell level on a large scale. Although IACS has been demonstrated for a variety of small-to-medium-size cells, applying it to large cells, cell clusters, and other large objects remains challenging, despite the scientific and industrial value in morphology-based sorting of such objects. The main difficulty lies in controlling large and complex cells throughout a microfluidic chip, from inlet to outlet especially the image acquisition and sorting regions. In particular, conventional IACS systems based on hydrodynamic focusing struggle to maintain stable focusing of large objects over the intervals required for processing images, leading to reduced sorting purity, yield, or event rate. To address these limitations, here we report an IACS system based on elasto-inertial focusing which enables IACS of large cells at high flow speeds of ∼1 m s^-1. We validated our developed elasto-inertial focuser by demonstrating that particles with a large diameter of >20 μm maintained their positions in the center of the focuser over a long distance of ≥35 mm. We integrated the focuser into the IACS system and sorted size-mixed particles (50% target) using a convolutional neural network-based classifier, demonstrating 96.0% purity and 80.5% yield at an event rate of 172 events per second (eps). Finally, we realized elasto-inertial focusing-based IACS of Euglena gracilis, a large microalgal cell species, based on intracellular lipid droplet formation, demonstrating 4.5-fold enrichment of target cells from 11.9% to 53.8% at 128 eps. Our work highlights that IACS based on elasto-inertial focusing enables the sorting of large objects based on high-content real-time image analysis without compromising sorting purity, yield, or event rate.