RT Journal Article
SR Electronic
T1 Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.11.24.469882
DO 10.1101/2021.11.24.469882
A1 Prakrit V. Jena
A1 Mitchell Gravely
A1 Christian C. Cupo
A1 Mohammad M. Safaee
A1 Daniel Roxbury
A1 Daniel A. Heller
YR 2021
UL http://biorxiv.org/content/early/2021/11/25/2021.11.24.469882.abstract
AB 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 diverse optical properties of a 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 nanomaterial emitters, to estimate nanomaterial counts in live cells and sub-cellular 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 to identify an upper-limit of the rate of uptake into cells - approximately 3,000 particles endocytosed within 30 minutes. In contrast, conventional ROI 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 heterogenous 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 cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for nanotoxicology, drug/gene delivery, and nanosensor fields.Competing Interest StatementD.A.H. is co-founder and officer with an equity interest in Goldilocks Therapeutics Inc., Lime Therapeutics Inc., and Nirova Biosense Inc. and a member of the scientific advisory boards of Concarlo Holdings LLC, Nanorobotics Inc., and Mediphage Bioceuticals Inc. P.V.J. is a co-founder with an equity interest in Lime Therapeutics, Inc.