Abstract
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.
Highlights
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; the currently available methods are limited in their labelling efficiency, specificity and cell permeability
Owing to the described limitations of existing labelling and transduction technologies, there is a persistent demand for techniques enabling high-throughput in-cell labelling by minimal tags that are conductive to high-resolution and super-resolution microscopy
We first investigated the specificity of the tris-N-nitrilotriacetic acid (trisNTA)/His tag targeting in chemically arrested cells
Summary
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; the currently available methods are limited in their labelling efficiency, specificity and cell permeability. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. Using the microfluidic cell squeezing platform to deliver small fluorescent tris-N-nitrilotriacetic acid (trisNTA) probes (B1 kDa), we demonstrate highly efficient, minimally disruptive, light-triggered tracing of native proteins and the subsequent super-resolution imaging of live-cell phenomena. The minimal size of the tag and the molecular probe allows direct targeting with nanometre precision at subnanomolar concentrations as required for single-molecule localization-based imaging techniques[1,2,12,13] with no impact on intracellular trafficking or demand for additional cofactors affecting endogenous processes. This facilitates fast uptake of probes into the cytosol before cell-intrinsic repair mechanisms kick in[15]
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