Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

Area of Science:

• Synthetic Biology
• Biophysics
• Cellular Engineering

Background:

• Synthetic cells are being developed by reconstituting cellular functions in liposomes.
• Microscopy and flow cytometry are key tools for characterizing liposomes, but have limitations.
• Current methods offer either detailed imaging or population statistics, not both simultaneously.

Purpose of the Study:

• To introduce imaging flow cytometry (IFC) for high-throughput screening of gene-expressing liposomes.
• To develop a comprehensive pipeline and analysis toolset for IFC in synthetic cell research.
• To overcome the limitations of separate microscopy and flow cytometry techniques.

Main Methods:

• Utilized a commercial imaging flow cytometry instrument and software.
• Developed a pipeline for analyzing ~60,000 liposome events per run from 1 µL stock solution.
• Quantified liposome phenotypes using fluorescence and morphological parameters from individual images.

Main Results:

• Achieved high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow.
• Enabled robust population statistics by analyzing individual liposome images.
• Quantified complex liposomal phenotypes relevant for synthetic cell construction.

Conclusions:

• Imaging flow cytometry (IFC) effectively integrates imaging and statistical analysis for liposome characterization.
• The developed pipeline and toolset facilitate the study of gene-expressing liposomes.
• IFC shows significant potential for advancing synthetic cell research, with discussed applicability and future prospects.