Open Access

Alina Klein, Susanne Hank, Anika Raulf, Eike F. Joest, Felicitas Tissen, Mike Heilemann, Ralph Wieneke, Robert Tampé

• Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.