Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells

The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise between information-rich imaging by microscopy and statistical population analysis by flow cytometry. To address this shortcoming, we here introduce imaging flow cytometry (IFC) for high-throughput, microscopy based screening of gene-expressing liposomes in laminar flow. We developed a comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software. About 60 thousands of liposome events were collected per run starting from one microliter of the stock liposome solution. Robust population statistics from individual liposome images was performed based on fluorescence and morphological parameters. This allowed us to quantify complex phenotypes covering a wide range of liposomal states that are relevant for building a synthetic cell. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are finally discussed.

Automated summary

The reconstitution of basic cellular functions in micrometer-sized liposomes has sparked interest in constructing synthetic cells. Microscopy and flow cytometry have limitations when used separately to study biological processes in liposomes, requiring a compromise between information-rich imaging and statistical analysis. To overcome this, imaging flow cytometry (IFC) is introduced for high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow. A comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software were developed, allowing for robust population statistics and quantification of complex phenotypes relevant for synthetic cell construction. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are discussed.