SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating serial and parallel sorting principles for accurate multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.