RT Journal Article
SR Electronic
T1 Multiplexed live-cell visualization of endogenous proteins with nanometer precision by fluorobodies
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 145698
DO 10.1101/145698
A1 Alina Klein
A1 Susanne Hank
A1 Anika Raulf
A1 Felicitas Tissen
A1 Mike Heilemann
A1 Ralph Wieneke
A1 Robert Tampé
YR 2017
UL http://biorxiv.org/content/early/2017/06/02/145698.abstract
AB The visualization of endogenous proteins in living cells is a major challenge. A fundamental requirement for spatiotemporally precise imaging is a minimal disturbance of protein function at high signal-to-background ratio. Current approaches for visualization of native proteins in living cells are limited by dark emitting, bulky fluorescent proteins and uncontrollable expression levels. Here, we demonstrate the labeling of endogenous proteins using nanobodies with site-specifically engineered bright organic fluorophores, named fluorobodies. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing allowed for low background, low toxicity, and high-throughput. Multiplexed imaging of distinct cellular structures was facilitated by specific protein targeting, culminating in live-cell super-resolution imaging of protein networks. The high-throughput delivery of engineered nanobodies will open new avenues in visualizing native cellular structures with unprecedented accuracy in cell-based screens.