Biology subjects

Gravely, M. A.

Publications and source records attributed to Gravely, M. A..

3 recordsLinked to original sources

bioRxiv2021

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 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.

bioengineering↗

bioRxiv2020

Multispectral Fingerprinting Resolves Dynamics of Nanomaterial Trafficking in Primary Endothelial Cells

Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials which gain cellular entry via active endocytic processes. A recent emphasis on routes of nanomaterial uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual role - that of an engineered nanomaterial (ENM) undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the unique optical properties exhibited by non-covalent hybrids of single-stranded DNA and SWCNTs (DNA-SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA-SWCNTs were observed to scale with increasing time after internalization. These were confirmed to be consequences of intracellular processes using pharmacological inhibitors of endosomal maturation, which suppressed spectral changes through two distinct mechanisms. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for single-chirality component spectra to be obtained with sub-micron spatial resolution. This approach uncovered a complex relationship between DNA-SWCNT concentration, fluorescence intensity, environmental transformations, and irreversible aggregation resulting from the temporal evolution of trafficking events. Finally, a spectral clustering analysis was applied to delineate the dynamic sequence of processes into four distinct populations, allowing stages of the intracellular trafficking process to be identified by the multispectral fingerprint of encapsulated DNA-SWCNTs. TOC Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗

bioRxiv2020

A Wearable Optical Microfibrous Biomaterial with Encapsulated Nanosensors Enables Wireless Monitoring of Oxidative Stress

In an effort to facilitate personalized medical approaches, the continuous and noninvasive monitoring of biochemical information using wearable technologies can enable a detailed understanding of an individuals physiology. Reactive oxygen species (ROS) are a class of oxygen-containing free radicals which function in a wide range of biological processes. In wound healing applications, the continuous monitoring of ROS through a wearable diagnostics platform is essential for the prevention of chronicity and pathogenic infection. Here, a versatile one-step procedure is utilized to fabricate optical core-shell microfibrous textiles incorporating single-walled carbon nanotubes (SWCNTs) for the real-time optical monitoring of hydrogen peroxide concentrations in wounds. The environmentally sensitive and non-photobleachable fluorescence of SWCNTs enables continuous analyte monitoring without a decay in signal over time. The existence of multiple chiralities of SWCNTs emitting near-infrared fluorescence with narrow bandwidths allows a ratiometric signal readout invariant to the excitation source distance and exposure time. The individual fibers encapsulate the SWCNT nanosensors for at least 21 days without apparent loss in structural integrity. Moreover, the microfibrous textiles can be utilized to spatially resolve peroxide concentrations on a wound surface using a camera and can be integrated into commercial wound bandages without being altered or losing their optical properties.

bioengineering↗