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.

Techniques based on fluorescence microscopy are increasingly used to count proteins in cells, but few stoichiometrically well-defined standards are available to test their accuracy. A selection of bacterial homo-oligomers were developed that contain 10-24 subunits and fully assemble when expressed in mammalian cells, and they can be used to easily validate/calibrate molecular counting methods. The utility of these standards was demonstrated by showing that nuclear pores contain 32 copies of the Nup107 complex.

We demonstrate super-resolution imaging of proteins and nucleic acids that were densely labelled with fluorophores using the concept of “click chemistry”.

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).

Using engineered nanobodies with bright organic dyes (fluorescent nanobodies) and subsequent microfluidic cell manipulation, controlled nanobody delivery was achieved, allowing the multiplexed imaging and super-resolution of endogenous protein networks in living cells.

Techniques based on fluorescence microscopya re increasingly used to count proteins in cells,b ut few stoichiometrically well-defined standards are available to test their accuracy.Aselection of bacterial homo-oligomers were developed that contain 10-24 subunits and fully assemble when expressed in mammalian cells,a nd they can be used to easily validate/calibrate molecular counting methods.T he utility of these standards was demonstrated by showing that nuclear pores contain 32 copies of the Nup107 complex.

Protein labeling with synthetic fluorescent probes is akey technology in chemical biology and biomedical research. As ensitive and efficient modular labeling approach (SLAP) was developed on the basis of as ynthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His 6-10 -tag.H igh-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques

A Small Labeling Pair (SLAP) for single‐molecule imaging techniques is reported by M. Heilemann, R. Tampé, and co‐workers in their Communication on The small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein (His6‐10‐tag) enable rapid, site‐specific, and stoichiometric labeling with high density. The proximity achieved with SLAP accounts for the higher spatial resolution with nanoscopy (orange) compared to wide‐field imaging (gray).