Show your true color: Mammalian cell surface staining for tracking cellular identity in multiplexing and beyond

Fluorescence microscopy revolutionized cell biology and changed requirements for dyes towards higher brightness, novel capacities, and specific targets. With the need for multiplexing assays in high-throughput methodologies, surface staining gained particular interest because it allows rapid application of exogenous stains to track cellular identity in mixed populations. Indeed, the last decade has enriched the toolbox of general lipid stains, fluorescent lipid analogues, sugar-binding lectins, and protein-specific antibodies enabling the first rationally designed plasma membrane-specific dyes. Still, multiple challenges exist, and the unique properties of each dye must be considered when selecting a staining approach for a specific application. Recent advances are also promising that future dyes will provide ultimate brightness and photostability in diverse colors and reduced sizes for high-resolution imaging.