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Fluorescence-activated cell sorting was first developed 52 years ago. Since then, the ability to isolate cells according to the expression of labeled proteins has revolutionized biological research. However, isolating individual cells with complex phenotypes is a technological challenge.

Now researchers present a high-speed cell sorter that uses fluorescence imaging to enable genome-scale studies of complex phenotypes. The technique adds fluorescence imaging and image-based decisioning to sort individual cells at exceptionally high speed, based on the visual details of each cell and not solely on the type or quantity of biomarkers that are present. The new technology could greatly expand the phenotypic space accessible to cell sorting applications and pooled genetic screening and has the potential to transform immunology, cell biology, and genomics research and enable new cell-based therapeutic discovery.

This work is published in Science, in the paper, “High speed fluorescence image-enabled cell sorting.

Single cells show a diversity of phenotypes, ranging from variable gene expression levels, dynamic protein localization, or differing cellular morphology. However, current approaches to cell sorting and characterization are limited spatially and lack subcellular resolution.

Schematic of image-enabled cell sorting, developed at BD Biosciences and road-tested by EMBL.
[BD Biosciences]

Daniel Schraivogel, PhD, research staff scientist at EMBL, and colleagues, combined “ultrafast microscopy and image analysis with a flow cytometric cell sorter to unlock spatial phenotypes for high-throughput sorting applications.” In doing so, they present a fully integrated high-speed image-enabled cell sorter (ICS), which records multicolor fluorescence images and sorts cells based on them at speeds upwards of 15,000 events per second.

The new innovation, known as the BD CellView Image Technology, can capture multiple images of individual cells flowing through the system and also adds a previously impossible capability of sorting cells based on detailed microscopic image analysis of individual cells at this speed.

By adding imaging to the traditional biomarker identification and quantification, the new technology not only identifies if and how much of a biomarker is present in the cell, but also its location or how it is distributed within the cell.

The researchers demonstrated ICS’s ability to rapidly isolate and quantify cells with complex cellular phenotypes, including cells with differently localized proteins and cells in different mitotic stages. They combined ICS with CRISPR-pooled screens to identify regulators of a nuclear factor pathway, nuclear factor κB (NF-κB), enabling the completion of genome-wide image-based screens in roughly nine hours of run time.

“This innovation has overcome the typical compromise between speed and precision of sorting individual cells,” said Tom Polen, chairman, CEO, and president of BD.

“For years, researchers have desired a system for cell sorting that would allow them to get a detailed picture of a cell’s inner workings and to isolate those with microscopic phenotypes of interest,” said Lars Steinmetz, PhD, senior scientist at EMBL and a professor of genetics at Stanford University. “This is what BD CellView Image Technology achieves, defining a new standard in cell isolation and characterization. We are excited about applying this technology to high-resolution genomic screening aimed at collecting functional information for every part of the genome. We are also exploring applications for cell-based diagnostics and characterization of cells in health and disease.”

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