Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles

Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the distinct optical properties of a heterogeneous nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed hyperspectral counting, which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of fluorescent nanomaterials to estimate particle number counts in live cells and subcellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique with single-walled carbon nanotubes and identified an upper limit of the rate of uptake into cells-approximately 3,000 nanotubes endocytosed within 30 min. In contrast, conventional region-of-interest counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogeneous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify the cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for the nanotoxicology, drug/gene delivery, and nanosensor fields.

Automated summary

This study proposes a method to improve quantification of nanomaterials at the single-cell and organelle level using the distinct optical properties of a heterogeneous nanomaterial preparation. The method, called hyperspectral counting, utilizes diffraction-limited imaging via hyperspectral microscopy and mathematical modeling to accurately estimate particle number counts in live cells and subcellular structures. The researchers applied this method to study the uptake of single-walled carbon nanotubes into cells and identified a higher rate of uptake compared to conventional counting methods. The study reveals significant heterogeneity and non-Gaussian distribution of nanotube uptake within cells, providing insights into cellular and subcellular nanoparticle concentrations.