Ultrasmall, Bright and Photostable Probes for Live-Cell Optical Super-Resolution Microscopy Based Interrogation of Complex Biological Processes

The development of ultrasmall optical probes that interrogate complex biological processes and states in situ and at the near-atomic scales without substantially perturbing the biological environment has been a long-standing goal. In this contribution we will introduce a particular class of ultrasmall (diameters below 7-8 nm) fluorescent silica core – poly(ethylene glycol) (PEG) shell (core-shell) nanoparticles referred to as Cornell dots (C dots) that allow live-cell optical super-resolution microscopy (SRM) in the form of stochastic optical reconstruction microscopy (STORM). Introduction of aluminum in the form of aluminosilicate cores, which covalently encapsulate fluorescent dyes chosen from a range of fluorophore families with absorption/emission wavelengths ranging from the optical into the near-infrared (NIR) regime, leads to optical blinking with low duty cycles ideal for STORM. These blinking processes, likely induced by redox mechanisms within the microporous aluminosilicate core, allow STORM experiments with a single excitation source and in the absence of cytotoxic imaging buffers, enabling live-cell SRM imaging. Live-cell STORM experiments on individual cells allow characterization, e.g. of the processing of such nanoprobes by cells via endocytotic pathways. Quantitative analysis of such STORM data sets provides information about the number of probes per intracellular vesicle as well as vesicle size. Furthermore, addition of a second sensor dye on the surface of such nanoprobes enables STORM-enhanced ratiometric sensing of metabolic analytes. Since these approaches can be generalized across a range of nanoprobe functionalities, including specific color, molecular targeting, payload delivery, and metabolic parameter sensing, the work provides a powerful nanomaterials platform for the interrogation of biological processes at unprecedented spatial resolution and with high molecular specificity in live-cell environments.