Real-time pooled optical screening with single-cell isolation capability

In a pooled optical screen, a genetically diverse library of living cells is imaged and characterised for phenotypic variations without knowing the genotype of the cells. The genotypes are identified in situ after the cells have been fixed or by physical extraction of interesting phenotypes followed by sequencing. Mother-machine microfluidics devices are efficient tools in pooled optical screens since many strains can be imaged in the same field of view, but the throughput is often limited. In this work, we show a method to extract single bacterial cells from a compact 100,000-trap mother-machine-based fluidic device using an optical tweezer. Unlike previous devices, the fluids in our design are routed in 3D to enable fast loading of cells, increased trap density, and faster imaging. We have also developed software that allows real-time analysis of the phenotyping data.