High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the FluoroDOT assay,'' which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-tonoise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.

Automated summary

The study introduces a method called FluoroDOT assay, which utilizes ultrabright nanoparticle plasmonic-fluors for high-resolution imaging of protein secretion. This versatile assay allows for exploration of spatial distribution of proteins secreted by single cells, the study of cell-to-cell heterogeneity in secretion, and detection of low abundance or incipient secretion of proteins.