Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles

Abstract

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.

Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes

“…Due to the optical diffraction limit of our system (∼500 nm) and the pixel size of our detector (∼150 nm), emission from a single SWCNT is blurred across a minimum of 3 × 3 pixels in NIR fluorescence images, and thus, small bundles and aggregates of SWCNTs could not be resolved from the single-nanotube population using Feret’s diameter measurements. Therefore, we employed a spectral counting method to investigate the degree of aggregation for each sample . Hyperspectral cubes of the spin-coated DNA–SWCNTs were acquired and again split into fluorescent ROIs.…”

“…Next, we sought to investigate how the intracellular processing of DNA–SWCNTs can vary depending on the dispersion quality. As a quantitative measure of this processing, the endosomal loading of DNA–SWCNTs was interrogated using the same hyperspectral counting approach previously described within single intracellular ROIs of the three cell lines . Hyperspectral cubes were acquired at 100× magnification for each cell type following a 30 min pulse of 250k-SWCNTs or 1k-SWCNTs and an additional 30 min of incubation in fresh media.…”

“…We utilized hyperspectral counting, a recently developed quantitative technique, to estimate the number of SWCNTs contained in diffraction-limited ROIs. Briefly, hyperspectral images were acquired at 100× magnification, initially processed to correct for background intensities, and split into individual ROIs corresponding to localized fluorescence from one or more nanotubes.…”

Nucleolin recognizing silica nanoparticles inhibit cell proliferation by activating the Bax/Bcl-2/caspase-3 signalling pathway to induce apoptosis in liver cancer

“…In particular, accumulating evidence has suggested that mesoporous silica nanoparticles (MSNs) play a vital role in drug delivery systems due to their low toxicity, superior biocompatibility, a large pore volume and surface area, and simplicity of functionalization ( Chen et al, 2022 ; Wei et al, 2022 ). Thus, MSNs have been widely utilized to load DNA ( Lin-Shiao et al, 2022 ; Ma et al, 2022 ), drugs ( Song et al, 2022 ; Zhang et al, 2022 ) and light ( Semcheddine et al, 2021 ; Jena et al, 2022 ) for cancer therapy and diagnosis. Furthermore, in order to increase the targeting efficiency, nanoparticles modified with a DNA or RNA aptamer are considered to be more promising owing to their low immunogenicity, smaller size, and active targeted property.…”

Super‐Resolution Near‐Infrared Fluorescence Microscopy of Single‐Walled Carbon Nanotubes Using Deep Learning

“…[8,9] Single-walled carbon nanotubes (SWCNTs), visualized as a 1D cylinder of a rolled-up sheet of carbon atoms arranged in a honeycomb lattice, find numerous biomedical applications, including imaging and sensing in the nIR. [10][11][12][13][14][15] With an inner diameter of 1-2 nm and length of 300 nm -10 μm, [16] SWCNTs have unique physical, chemical, mechanical, and electronic properties. In particular, SWCNTs emit bright fluorescence in the nIR range (900-1400 nm), which does not photobleach nor blink.…”