Protein nanobarcodes enable single-step multiplexed fluorescence imaging

Author:

de Jong-Bolm DaniëlleORCID,Sadeghi MohsenORCID,Bogaciu Cristian A.,Bao Guobin,Klaehn Gabriele,Hoff Merle,Mittelmeier Lucas,Basmanav F. Buket,Opazo Felipe,Noé Frank,Rizzoli Silvio O.

Abstract

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.

Funder

European’s Union Horizon 2020 Horizon research and innovation program

Deutsche Forschungsgemeinschaft

European Research Commission

Bundesministerium für Bildung und Forschung

Berlin Institute for Foundations in Learning and Data

Campus Labaratory for Advanced Imaging, Microscopy and Spectroscopy

Publisher

Public Library of Science (PLoS)

Subject

General Agricultural and Biological Sciences,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience

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