Abstract: The significance of lipid droplets in lipid metabolism, cell signaling, and regulating longevity is increasingly recognized, yet the lipid droplet’s unique properties and architecture make it difficult to size and study using conventional methods. To begin to address this issue, we demonstrate the capabilities of nanoparticle tracking analysis (NTA) for sizing of lipid droplets. NTA was found to be adequate to assess lipid droplet stability over time, indicating that lipid droplet preparations are stable for up to 24 h. NTA had the ability to compare the size distributions of lipid droplets from adult and geriatric mouse liver tissue, suggesting an age-related decrease in lipid droplet size. This is the first report on the use of NTA to size intracellular organelles.

Abstract: Exosomes are extracellular vesicles of 30 to100 nm in diameter, which are secreted by various cells of mammals. It is known that exosomes represent a specific way of cell-to-cell communication. Mechanisms of secretion and uptake of these vesicles are not clear. To address this problem, we fused the transmembrane protein CD63, a known exosomal marker, at its N-termini to the EGFP protein. We have shown that these proteins exit cells as a part of exosomes, substantiated by the fluorescent nanoparticle tracking analysis. The analysis has also shown that there are two prevailing exosome populations in general population of vesicles.

Abstract: Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication, and in mammals are generally classified as ~50-150nm exosomes, ~100-1000nm microvesicles, and apoptotic bodies, each arising as a result of different biological processes. EVs carry protein, lipids, and nucleic acids within the circulation, to target cells whereupon they mediate physiological changes. Due to their small size, quantification and characterization by conventional microscopy is not possible. However, nanoparticle tracking analysis (NTA) has provided a method to determine the fluid concentration and size of extracellular vesicles and other nanoparticles. While NTA provides statistical summaries of samples in an experiment, a recurring difficulty is the organization, manipulation, and management of raw particle count data because of the large size of datasets, and resultant vulnerability to user error. To address these limitations, we developed tidyNano, an R package that provides functions to import, clean, and quickly summarize raw NanoSight (Malvern Panalytical) data for efficient calculation of statistics and visualization. Here, we provide a framework for importing raw nanoparticle data and provide functions to facilitate rapid and efficient analysis, visualization and calculation of summary statistics. tidyNano was used to analyze murine plasma extracellular vesicles across gestation by aggregating and summarizing samples based on technical, biological and gestational parameters. In addition, we developed shinySIGHT, a Shiny web application that allows for interactive exploration and visualization of EV data. Using this package, we analyzed data generated from 36 samples of EV derived from the plasma of mice across gestation. Peripheral EV concentration increased linearly across pregnancy, with trending increases as early as gestation day (GD) 5.5 and significant rises at GD14.5, and 17.5 relative to EV concentrations in nonpregnant females. Thus, the data highlight the utility of the mouse as a model of EV biology in pregnancy. Further, the package provides a mechanism for seamless analysis of EV data generated by NanoSight. Importantly, this package provides a straightforward framework by which diverse types of large datasets can be simply and efficiently analyzed. tidyNano and shinySIGHT are freely available under the MIT license and is hosted on GitHub (https://nguyens7.github.io/tidyNano/).

Abstract: The experimental setup for determination of particle sizes by nanotracking is developed. It is shown the possibility to identify particles with different fluorescent labels using developed experimental setup. The first results of analysis for samples of polystyrene micro- and nanoparticles are obtained using developed experimental setup.

Abstract: Methods to efficiently measure the concentration of nanoparticles in liquid suspensions are extremely important in nanoparticle-based applications. Existing nanoparticle concentration measurement methods such as nanoparticle tracking analysis (NTA), single particle-inductively coupled plasma-mass spectroscopy (SP-ICP-MS) and TEM have inherent limitations, including size requirements, special physical or chemical properties and special sample preparation. This paper presents a novel method to measure the concentration of nanoparticles by atomic force microscope. Nanoparticles distribution property after complete evaporation on silicon wafer was found and particles amount estimation theory was established, which enabled accurate and efficient measurement of nanoparticles concentration. The nanoparticle suspensions with different concentration, size and type were measured by the proposed approach, respectively. The results were compared with the estimated concentration by mass fraction, which demonstrated the effectiveness and correctness of this AFM-based measurement method.

Abstract: Extracellular vesicles (EVs) are now intensively studied in the context of their action on the irradiated organism. Suspensions of EVs isolated from culture media of two different cell lines were analyzed by transmission electron microscopy and nanoparticle tracking analysis (NTA) to evaluate size and concentration of contained particles. The results obtained indicate the difference in analyzed parameters depending on the cell type. Keywords: stem cells, extracellular vesicles, nanoparticle tracking analysis

Abstract: The experimental setup for multispectral nanoparticle tracking analysis is developed. The optical system for sample illumination in nanoparticle tracking analysis is designed that makes it possible to obtain images of particles in suspensions with a high signal-to-noise ratio. The results of the comparison of particle size distributions obtained by nanoparticle tracking analysis using developed experimental setup, dynamic light scattering method and scanning electron microscope are shown.

Abstract: Figure S1. Characterization of hMSC-derived exosomes isolated by using a total exosome isolation kit. a Representative NTA graph plots depicting the number of exosomes secreted by tissue-specific hMSCs, which showed that BMSCs secrete a higher number of exosomes in comparison with ADSCs within 48 h. b Size distribution graph plots for hMSCs showed that, as measured by NTAs, the particle size for hMSC exosomes isolated from both BMSCs and ADSCs was within the range of 30 to 120 nm. c Transmission electron microscopic pictures of exosomes isolated by hMSCs showed cup-shaped morphology of exosomes. Results are mean Âą standard error of the mean of three independent experiments. *Significant with P value of less than 0.05. Abbreviations: ADSC adipose tissueâ derived mesenchymal stem cell, BMSC bone marrowâ derived mesenchymal stem cell, hMSC human mesenchymal stem cell, NS non-significant, NTA Nanoparticle Tracking Analysis (PDF 217 kb)

Abstract: The relative scarcity of well-defined genetic and metabolic linkages to material properties impedes biological production of inorganic materials. The physiology of electroactive bacteria is intimately tied to inorganic transformations, which makes genetically tractable and well-studied electrogens, such as <i>Shewanella oneidensis,</i> attractive hosts for material synthesis. Notably, this species is capable of reducing a variety of transition-metal ions into functional nanoparticles, but exact mechanisms of nanoparticle biosynthesis remain ill-defined. We report two key factors of extracellular electron transfer by <i>S. oneidensis</i>, the outer membrane cytochrome, MtrC, and soluble redox shuttles (flavins), that affect Pd nanoparticle formation. Changes in the expression and availability of these electron transfer components drastically modulated particle synthesis rate and phenotype, including their structure and cellular localization. These relationships may serve as the basis for biologically tailoring Pd nanoparticle catalysts and could potentially be used to direct the biogenesis of other metal nanomaterials.

Abstract: Figure S1. Characterization and quantification of exosome levels and heatmap of the up-regulated exosomal lncRNAs and mRNAs identified in the screening. (A) Transmission electron microscopy (TEM) revealed the phenotype of exosomes isolated from stage I GC patient and healthy control plasma samples. Scale bar, 100 nm. (B) TEM revealed the phenotype of exosomes isolated from GCC and HPSEC culture media. Scale bar, 200 nm. (C and D) NanoSight particle tracking analysis of the size distributions and number of exosomes isolated from stage I GC patient and healthy control plasma samples (C) and isolated from GCC and HPSEC culture media (D). (E and F) Western blot showing the presence of specific exosome marker proteins (CD9 and CD63) and the absence of tubulin in exosomes derived from stage I GC patient and healthy control plasma samples (E) and derived from GCC and HPSEC culture media (F). (G) Heatmap of the 79 up-regulated exosomal lncRNAs and mRNAs between the plasma samples of stage I GC patients (n = 10) and healthy controls (n = 5). (H) Heatmap of the 285 up-regulated exosomal lncRNAs and mRNAs between GCC (n = 4) and HPSEC (n = 4) culture media. (PDF 395 kb)