Abstract
The interrogation of single cells has revolutionised biology and medicine by providing crucial unparalleled insights into cell-to-cell heterogeneity. Flow cytometry (including fluorescence-activated cell sorting) is one of the most versatile and high-throughput approaches for single-cell analysis by detecting multiple fluorescence parameters of individual cells in aqueous suspension as they flow past through a focus of excitation lasers. However, this approach relies on the expression of cell surface and intracellular biomarkers, which inevitably lacks spatial and temporal phenotypes and activities of cells, such as secreted proteins, extracellular metabolite production, and proliferation. Droplet microfluidics has recently emerged as a powerful tool for the encapsulation and manipulation of thousands to millions of individual cells within pico-litre microdroplets. Integrating flow cytometry with microdroplet architectures surrounded by aqueous solutions (e.g., water-in-oil-in-water (W/O/W) double emulsion and hydrogel droplets) opens avenues for new cellular assays linking cell phenotypes to genotypes at the single-cell level. In this review, we discuss the capabilities and applications of droplet flow cytometry (DFC). This unique technique uses standard commercially available flow cytometry instruments to characterise or select individual microdroplets containing single cells of interest. We explore current challenges associated with DFC and present our visions for future development.
Highlights
IntroductionSingle-cell analysis plays an essential role in revealing the differences of individual cells in structure and function.[1,2] The adaption of single-cell techniques has highlighted critical cellto-cell heterogeneity, identi ed rare sub-populations of functional importance and discovered unique characteristics of individual cells.[3,4] Flow cytometry (FC) has been widely used as a versatile and powerful technique to interrogate single cells, offering automated, quantitative, high-throughput and multiparameter characterisation of cell phenotypes and activities.[5,6] In this technique, individual cells suspended in a uid ow are directed to pass through a laser beam one by one and measured based on the resulting scatter or emission of light energy from uorescence molecules
Unlike conventional water-in-oil (W/O) droplets, two main architectures of droplets suspended in aqueous solutions are compatible with Flow cytometry (FC): water-in-oil-inwater (W/O/W)[18] double emulsion (DE) droplets and hydrogel droplets.[19]
The pairing of FC and droplets allows a complete characterisation of individual cells beyond the conventional surface and intracellular biomarkers, featuring high throughput, sensitivity and dynamic range, and low cost
Summary
Single-cell analysis plays an essential role in revealing the differences of individual cells in structure and function.[1,2] The adaption of single-cell techniques has highlighted critical cellto-cell heterogeneity, identi ed rare sub-populations of functional importance and discovered unique characteristics of individual cells.[3,4] Flow cytometry (FC) has been widely used as a versatile and powerful technique to interrogate single cells, offering automated, quantitative, high-throughput and multiparameter characterisation of cell phenotypes and activities.[5,6] In this technique, individual cells suspended in a uid ow are directed to pass through a laser beam one by one and measured based on the resulting scatter or emission of light energy from uorescence molecules. Unlike conventional water-in-oil (W/O) droplets, two main architectures of droplets suspended in aqueous solutions are compatible with FC: water-in-oil-inwater (W/O/W)[18] double emulsion (DE) droplets and hydrogel droplets.[19] The pairing of FC and droplets allows a complete characterisation of individual cells beyond the conventional surface and intracellular biomarkers, featuring high throughput, sensitivity and dynamic range, and low cost It enables tandem genomic, epigenomic, or transcriptomic analyses on isolated cells,[20] allowing integrative single-cell analysis techniques. Different types of single cells (e.g., mammalian, bacterial and yeast cells) are encapsulated individually within microdroplets (either W/O/W DE droplets or hydrogel droplets), which can be screened and sorted by commercial FC machines, and further analysed at the downstream (e.g., sequencing) This integrated technology, so called droplet ow cytometry (DFC), allows a variety of biochemical assays at the single-cell level, such as single-cell cultivation, molecular evolution, single-cell detection and cell-to-cell interactions, due to droplet monodispersity, cell compartmentalisation and high-throughput processing. The isolated cells from heterogeneous populations allow downstream bioinformatics analysis (e.g., tandem genomic, epigenomic and transcriptomic analyses) of the same target, revealing the biological functions of massive genetic pathways.[48,49,50]
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