Abstract
In the replacement of genetic probes, there is increasing interest in labeling living cells with high-quality extrinsic labels, which avoid over-expression artifacts and are available in a wide spectral range. This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells. Here, we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique. We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation. We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses, allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells. We demonstrate high-quality time-lapse imaging with confocal, total internal reflection fluorescence (TIRF), and Airyscan super-resolution microscopy. As the entire procedure is readily compatible with fluorescence (super resolution) microscopy, photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.
Highlights
It is imperative to observe subcellular structures as well as intracellular processes to gain insight into the role of biomolecules and biological pathways[1]
To quantify more precisely how many vapor nanobubbles (VNBs) can be formed per graphene quantum dots (GQDs), we repeated the experiment for nanoparticles incubated with HeLa cells for 30 min, allowing them to bind to the cell membrane
Here, we have proposed VNB photoporation as a generic technology to enable live-cell labeling with exogenous labels for fluorescence microscopy
Summary
It is imperative to observe subcellular structures as well as intracellular processes to gain insight into the role of biomolecules and biological pathways[1]. While highquality organic and particulate labels are available for fluorescence (super resolution) microscopy, their use is mainly limited to fixed and permeabilized cells, as they cannot readily permeate through the cell membrane of living cells[2]. This is why genetic engineering with fluorescent proteins has become the predominant labeling method for live cells in the last 15 years. Several intracellular delivery methods have been evaluated for delivering extrinsic labels into live cells for microscopy.
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