Live-cell protein labelling with nanometre precision by cell squeezing

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.

Labeling proteins inside living cells using external fluorophores for microscopy

“…However, these techniques have significant drawbacks such as low throughput and the requirement of additional apparatus. Finally, two recently developed methods, such as ‘biophotonic laser-assisted surgery tool (BLAST)’ and ‘cell squeezing’ are promising techniques, although they require the cells to be cultured in specific platforms like fabricated surface or microfluidic channels (Wu et al, 2015; Kollmannsperger et al, 2016). …”

Survival rate of eukaryotic cells following electrophoretic nanoinjection

“…These are, in most cases, used on large numbers of cells in culture and it is commonly accepted that a significant number of these cells (up to 50%) will either not survive this process4 or that the cell cycle of a significant number of cells is disrupted5. Newer techniques such as cell squeezing67, or massive parallel delivery with light pulses8 enable more control over the process but are still of a stochastic nature. These stochastic processes lack the ability to specifically address single cells.…”

Multivalent Chelators for In Vivo Protein Labeling

“…After internalization, efficient and rapid intracellular targeting of Histagged proteins in mammalian cells was observed. [55] Here,m ammalian cells were mechanically pushed (squeezed) through defined micrometer constrictions in the presence of nanomolar concentrations of dye-conjugated trisNTA ( Figure 5a). His-tag-specific labeling was further monitored in real-time and demonstrated high specificity and ahigh degree of co-localization, even in the crowded cellular environment.…”