Protein nanobarcodes enable single-step multiplexed fluorescence imaging

Multiplexed cellular imaging typically relies on the sequential application of detection probes, as antibodies or DNA barcodes, which is complex and time-consuming. To address this, we developed here protein nanobarcodes, composed of combinations of epitopes recognized by specific sets of nanobodies. The nanobarcodes are read in a single imaging step, relying on nanobodies conjugated to distinct fluorophores, which enables a precise analysis of large numbers of protein combinations. Fluorescence images from nanobarcodes were used as input images for a deep neural network, which was able to identify proteins with high precision. We thus present an efficient and straightforward protein identification method, which is applicable to relatively complex biological assays. We demonstrate this by a multicell competition assay, in which we successfully used our nanobarcoded proteins together with neurexin and neuroligin isoforms, thereby testing the preferred binding combinations of multiple isoforms, in parallel. Multiplexed cellular imaging typically relies on the sequential application of detection probes, which is complex and time-consuming. This study describes a novel approach to protein labeling, in which individual proteins are tagged with combinations of multiple epitopes. This allows a normal four-color-channel microscope to reveal identities of up to fifteen different proteins, enabling simple and powerful multiplexing.