Recent Progress in Hair Science and Trichology.

Hair is important to our appearance as well as to protect our heads. Human hair mainly consists of proteins (80-85%), melanin pigments (0-5%), water (10-13%), and lipids (1-6%). The physicochemical properties of hair have been studied for over 100 years. However, they are not yet thoroughly understood. In this review, recent progress and the latest findings are summarized from the following three perspectives: structural characteristics, delivery and distribution of active ingredients, and hair as a template. The structural characteristics of hair have been mainly investigated by microscopic and/or spectroscopic techniques such as atomic force microscopy integrated with infrared spectroscopy (AFM-IR) and rheological measurements. The distribution of active ingredients has been generally evaluated through techniques such as nanoscale secondary ion mass spectrometry (NanoSIMS). And finally, attempts to explore the potential of hair to be used as a substrate for flexible device fabrication will be introduced.

A Hitchhiker's guide to high-dimensional tissue imaging with multiplexed ion beam imaging.

Advancements in multiplexed tissue imaging technologies are vital in shaping our understanding of tissue microenvironmental influences in disease contexts. These technologies now allow us to relate the phenotype of individual cells to their higher-order roles in tissue organization and function. Multiplexed Ion Beam Imaging (MIBI) is one of such technologies, which uses metal isotope-labeled antibodies and secondary ion mass spectrometry (SIMS) to image more than 40 protein markers simultaneously within a single tissue section. Here, we describe an optimized MIBI workflow for high-plex analysis of Formalin-Fixed Paraffin-Embedded (FFPE) tissues following antigen retrieval, metal isotope-conjugated antibody staining, imaging using the MIBI instrument, and subsequent data processing and analysis. While this workflow is focused on imaging human FFPE samples using the MIBI, this workflow can be easily extended to model systems, biological questions, and multiplexed imaging modalities.

ToF-SIMS imaging shows specific lipophilic vitamin alterations in chronic reprotoxicity caused by the emerging contaminant Pravastatin in Gammarus fossarum.

Blood lipid-lowering agents, such as Pravastatin, are among the most frequently used pharmaceuticals released into the aquatic environment. Although their effects on humans are very well understood, their consequences on freshwater organisms are not well known, especially in chronic exposure conditions. Gammarus fossarum is commonly used as sentinel species in ecotoxicology because of its sensitivity to a wide range of environmental contaminants and the availability of standardized bioassays. Moreover, there is an increased interest in linking molecular changes in sentinel species, such as gammarids, to observed toxic effects. Here, we performed a reproductive toxicity assay on females exposed to different concentrations of pravastatin (30; 300; 3,000 and 30,000 ng L-1) during two successive reproductive cycles and we applied ToF-SIMS imaging to evaluate the effect of pravastatin on lipid homeostasis in gammarids. Reproductive bioassay showed that pravastatin could affect oocyte development in Gammarus fossarum inducing embryotoxicity in the second reproductive cycle. Mass spectrometry imaging highlighted the disruption in vitamin E production in the oocytes of exposed female gammarids at the second reproductive cycle, while limited alterations were observed in other lipid classes, regarding both production and tissue distribution. The results demonstrated the interest of applying spatially resolved lipidomics by mass spectrometry imaging to assess the molecular effects induced by long-term exposure to environmental pharmaceutical residues in sentinel species.

Quantitative analysis of molecular interactions in κ-carrageenan-Isovanillin biocomposite for biodegradable packaging and pharmaceutical applications using NMR, TOF-SIMS, and XPS approach.

This study explores the molecular interactions and structural changes in κ-carrageenan crosslinked with isovanillin to create a biocomposite material suitable for hard capsule and bio-degradable packaging applications. Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy revealed chemical changes in the conjugate molecule, indicating improved electronegativity due to intermolecular hydrogen bonding between κ-carrageenan and isovanillin. Time-of-flight Secondary Ion Mass Spectrometry (ToF-SIMS) analysis revealed enhanced ion intensity due to intermolecular interactions, particularly between sulphate and hydrogen ions. X-ray Photoelectron Spectroscopy (XPS) study demonstrated that κ-carrageenan and isovanillin form stronger hydrogen bonds, with a shift in binding energy indicating higher electronegativity. These findings shed light on the molecular mechanisms that underpin the formation of the biocomposite material, as well as its potential for use in hard capsule and biodegradable packaging materials, addressing the need for sustainable alternatives in the pharmaceutical and packaging industries while also contributing to environmental conservation.

Stable isotopes and nanoSIMS single-cell imaging reveals soil plastisphere colonizers able to assimilate sulfamethoxazole.

The presence and accumulation of both, plastics and antibiotics in soils may lead to the colonization, selection, and propagation of soil bacteria with certain metabolic traits, e.g., antibiotic resistance, in the plastisphere. However, the impact of plastic-antibiotic tandem on the soil ecosystem functioning, particularly on microbial function and metabolism remains currently unexplored. Herein, we investigated the competence of soil bacteria to colonize plastics and degrade 13C-labeled sulfamethoxazole (SMX). Using single-cell imaging, isotope tracers, soil respiration and SMX mineralization bulk measurements we show that microbial colonization of polyethylene (PE) and polystyrene (PS) surfaces takes place within the first 30 days of incubation. Morphologically diverse microorganisms were colonizing both plastic types, with a slight preference for PE substrate. CARD-FISH bacterial cell counts on PE and PS surfaces formed under SMX amendment ranged from 5.36 × 103 to 2.06 × 104, and 2.06 × 103 to 3.43 × 103 hybridized cells mm-2, respectively. Nano-scale Secondary Ion Mass Spectrometry measurements show that 13C enrichment was highest at 130 days with values up to 1.29 atom%, similar to those of the 13CO2 pool (up to 1.26 atom%, or 22.55 ‰). Independent Mann-Whitney U test showed a significant difference between the control plastisphere samples incubated without SMX and those in 13C-SMX incubations (P < 0.001). Our results provide direct evidence demonstrating, at single-cell level, the capacity of bacterial colonizers of plastics to assimilate 13C-SMX from contaminated soils. These findings expand our knowledge on the role of soil-seeded plastisphere microbiota in the ecological functioning of soils impacted by anthropogenic stressors.

Resting cells of Skeletonema marinoi assimilate organic compounds and respire by dissimilatory nitrate reduction to ammonium in dark, anoxic conditions.

Diatoms can survive long periods in dark, anoxic sediments by forming resting spores or resting cells. These have been considered dormant until recently when resting cells of Skeletonema marinoi were shown to assimilate nitrate and ammonium from the ambient environment in dark, anoxic conditions. Here, we show that resting cells of S. marinoi can also perform dissimilatory nitrate reduction to ammonium (DNRA), in dark, anoxic conditions. Transmission electron microscope analyses showed that chloroplasts were compacted, and few large mitochondria had visible cristae within resting cells. Using secondary ion mass spectrometry and isotope ratio mass spectrometry combined with stable isotopic tracers, we measured assimilatory and dissimilatory processes carried out by resting cells of S. marinoi under dark, anoxic conditions. Nitrate was both respired by DNRA and assimilated into biomass by resting cells. Cells assimilated nitrogen from urea and carbon from acetate, both of which are sources of dissolved organic matter produced in sediments. Carbon and nitrogen assimilation rates corresponded to turnover rates of cellular carbon and nitrogen content ranging between 469 and 10,000 years. Hence, diatom resting cells can sustain their cells in dark, anoxic sediments by slowly assimilating and respiring substrates from the ambient environment.

Imaging with mass spectrometry: Which ionization technique is best?

The use of mass spectrometry (MS) to acquire molecular images of biological tissues and other substrates has developed into an indispensable analytical tool over the past 25 years. Imaging mass spectrometry technologies are widely used today to study the in situ spatial distributions for a variety of analytes. Early MS images were acquired using secondary ion mass spectrometry and matrix-assisted laser desorption/ionization. Researchers have also designed and developed other ionization techniques in recent years to probe surfaces and generate MS images, including desorption electrospray ionization (DESI), nanoDESI, laser ablation electrospray ionization, and infrared matrix-assisted laser desorption electrospray ionization. Investigators now have a plethora of ionization techniques to select from when performing imaging mass spectrometry experiments. This brief perspective will highlight the utility and relative figures of merit of these techniques within the context of their use in imaging mass spectrometry.

Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging.

Membrane lipids have been known to influence multiple signalling and cellular processes. Dysregulation of lipids at the neuronal membrane is connected to a significant alteration of the brain function and morphology, leading to brain diseases and neurodegeneration. Understanding the lipid composition and turnover of neuronal membrane will provide a significant insight into the molecular events underlying the regulatory effects of these biomolecules in a neuronal system. In this study, we aimed to characterize the composition and turnover of the plasma membrane lipids in human neural progenitor cells (NPCs) at an early differentiation stage into midbrain neurons using ToF-SIMS imaging. Lipid composition of the native plasma membrane was explored, followed by an examination of the lipid turnover using different isotopically labelled lipid precursors, including 13C-choline, 13C-lauric acid, 15N-linoleic, and 13C-stearic. Our results showed that differentiating NPCs contain a high abundance of ceramides, glycerophosphoserines, neutral glycosphingolipids, diradylglycerols, and glycerophosphocholines at the plasma membrane. In addition, different precursors were found to incorporate into different membrane lipids which are specific for the short- or long-carbon chains, and the unsaturation or saturation stage of the precursors. The lipid structure of neuronal membrane reflects the differentiation status of NPCs, and it can be altered significantly using a particular lipid precursor. Our study illustrates a potential of ToF-SIMS imaging to study native plasma membrane lipids and elucidate complex cellular processes by providing molecular -rich information at a single cell level.

Atomistic simulations for investigation of substrate and salt effects on lipid in-source fragmentation in secondary ion mass spectrometry: A follow-up study.

In-source fragmentation (ISF) poses a significant challenge in secondary ion mass spectrometry (SIMS). These fragment ions increase the spectral complexity and can lead to incorrect annotation of fragments as intact species. The presence of salt that is ubiquitous in biological samples can influence the fragmentation and ionization of analytes in a significant manner, but their influences on SIMS have not been well characterized. To elucidate the effect of substrates and salt on ISF in SIMS, we have employed experimental SIMS in combination with atomistic simulations of a sphingolipid on a gold surface with various NaCl concentrations as a model system. Our results revealed that a combination of bond dissociation energy and binding energy between N-palmitoyl-sphingomyelin and a gold surface is a good predictor of fragment ion intensities in the absence of salt. However, ion-fragment interactions play a significant role in determining fragment yields in the presence of salt. Additionally, the charge distribution on fragment species may be a major contributor to the varying effects of salt on fragmentation. This study demonstrates that atomistic modeling can help predict ionization potential when salts are present, providing insights for more accurate interpretations of complex biological spectra.

Targeted intradermal delivery of alpha-arbutin-loaded dissolving polymeric microneedles visualized by three-dimensional Orbitrap secondary ion mass spectrometry (3D OrbiSIMS).

Hyperpigmentation, a prevalent dermatological condition characterized by melanin overproduction, poses treatment challenges due to the hydrophilicity of alpha-arbutin, a widely utilized tyrosinase inhibitor. This study investigates the efficacy of dissolving microneedles (DMNs) in augmenting skin permeation for alpha-arbutin delivery to the targeted epidermal site. Porcine full-thickness skin was employed in a 24-hour Franz cell study, commencing with the assessment of commercial alpha-arbutin-containing products. Solid steel microneedles (CMNs) from Dermapen® were utilized as both pre- and post-treatment modalities to evaluate the influence of different applications on alpha-arbutin delivery. Additionally, alpha-arbutin-loaded polyvinylpyrrolidone-co-vinyl acetate (PVPVA) DMNs, containing 2 % w/w alpha-arbutin, were fabricated and examined for their permeation-enhancing capabilities. HPLC analysis and 3D Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) were employed to quantify and visualize alpha-arbutin in various Franz cell components. Results indicate that alpha-arbutin permeation to the skin was restricted (less than 1 %) without microneedle application and significantly increased by 6-fold (4-5 %) with post-treatment CMNs and DMNs, but not with pre-treatment CMNs. Notably, DMNs exhibited a more sustainable and robust capacity than post-treatment CMNs. OrbiSIMS imaging analysis revealed that DMNs visually enhance skin permeation of alpha-arbutin by delivering the compound to the basal layer of the targeted skin location. Overall, this study underscores the potential of DMNs as a promising delivery system for promoting targeted intradermal delivery of alpha-arbutin, providing a comprehensive exploration of various methodologies to identify innovative and improved microneedle approaches for alpha-arbutin permeation.

Monitoring lipid alterations in Drosophila heads in an amyotrophic lateral sclerosis model with time-of-flight secondary ion mass spectrometry.

Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.

High-Throughput Quantitative Analysis of Amino Acids in Freeze-Dried Drops Using Time-of-Flight Secondary Ion Mass Spectrometry.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has become a promising analytical tool for molecular profiling in biological applications. However, its ultrahigh vacuum environment and matrix effects hamper the absolute quantitation of solution samples. Herein, we present a rapid high-throughput platform for quantitative ToF-SIMS analysis of amino acids in matrix deposits formed from freeze-dried solution drops through ice sublimation on a parylene film microarray substrate. Droplets of the amino acid solutions, which were mixed with stable isotope-labeled phenylalanine (F*) of high concentration (10 mM), were loaded on wells of the microarray, then frozen and evaporated slowly below the freezing point, forming continuous solid-phase F* matrix deposits. The amino acids (≤500 µM), adequately well dispersed throughout the F* matrix deposits on each well, were quantitatively analyzed by ToF-SIMS in a rapid and high-throughput fashion. The lower limit of quantitation reached below 10 µM.

Leveraging ToF-SIMS imaging to investigate tenofovir disoproxil fumarate degradation at excipient interfaces in oral compressed tablets.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used to study the hydrolysis of tenofovir disoproxil fumarate (TDF) to tenofovir monosoproxil (TM) within an oral compressed tablet. The ToF-SIMS images displayed a heterogenous distribution of the matrix components. Evaluation of the TM distribution revealed that it was primarily co-localized with areas of higher excipient concentration pointing toward excipient driven degradation. To support these observations, a compatibility study of TDF with each tablet component was performed via liquid chromatography. The ToF-SIMS imaging and compatibility study indicated that the excipient, Avicel® PH-102, was the primary driver of TM formation in the tablet. The hydrolysis degradation mechanism within the tablet is further rationalized through discussion of chemical and physical properties of the matrix components. The sum of this work demonstrates a new analytical workflow for probing and understanding matrix driven degradation in oral compressed tablets utilizing ToF-SIMS imaging.

Comparison of δ13 C analyses of individual foraminifer (Orbulina universa) shells by secondary ion mass spectrometry and gas source mass spectrometry.

RATIONALE: The use of secondary ion mass spectrometry (SIMS) to perform micrometer-scale in situ carbon isotope (δ13 C) analyses of shells of marine microfossils called planktic foraminifers holds promise to explore calcification and ecological processes. The potential of this technique, however, cannot be realized without comparison to traditional whole-shell δ13 C values measured by gas source mass spectrometry (GSMS). METHODS: Paired SIMS and GSMS δ13 C values measured from final chamber fragments of the same shell of the planktic foraminifer Orbulina universa are compared. The SIMS-GSMS δ13 C differences (Δ13 CSIMS-GSMS ) were determined via paired analysis of hydrogen peroxide-cleaned fragments of modern cultured specimens and of fossil specimens from deep-sea sediments that were either untreated, sonicated, and cleaned with hydrogen peroxide or vacuum roasted. After treatment, fragments were analyzed by a CAMECA IMS 1280 SIMS instrument and either a ThermoScientific MAT-253 or a Fisons Optima isotope ratio mass spectrometer (GSMS). RESULTS: Paired analyses of cleaned fragments of cultured specimens (n = 7) yield no SIMS-GSMS δ13 C difference. However, paired analyses of untreated (n = 18) and cleaned (n = 12) fragments of fossil shells yield average Δ13 CSIMS-GSMS values of 0.8‰ and 0.6‰ (±0.2‰, 2 SE), respectively, while vacuum roasting of fossil shell fragments (n = 11) removes the SIMS-GSMS δ13 C difference. CONCLUSIONS: The noted Δ13 CSIMS-GSMS values are most likely due to matrix effects causing sample-standard mismatch for SIMS analyses but may also be a combination of other factors such as SIMS measurement of chemically bound water. The volume of material analyzed via SIMS is ~105 times smaller than that analyzed by GSMS; hence, the extent to which these Δ13 CSIMS-GSMS values represent differences in analyte or instrument factors remains unclear.

Characterization of Sulfur-Rich Microbial Organic Matter in Jurassic Carbonates Using Laser-Assisted Mass Spectrometry.

Laser desorption-ionization mass spectrometry (MS) shows great potential for in situ molecular analysis of planetary surfaces and microanalysis of space-returned samples or (micro)fossils. Coupled with pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) in ESA's ExoMars project, this technique could help assess further the origin of sulfur-bearing organic matter (OM) recently detected on Mars. To unravel this potential, we analyzed sulfurized microbial OM from ca. 150 million year-old carbonates with laser desorption-ionization mass spectrometry (single- and two-step: LDI-MS and L2MS), in comparison with time-of-flight secondary-ion mass spectrometry (ToF-SIMS), gas chromatography-mass spectrometry (GC-MS), and Py-GC-MS. We show that LDI-MS and L2MS readily detect sulfur-bearing moieties such as (alkyl)thiophenes and (alkyl)benzothiophenes. The mineral matrix, however, made the identification of sulfur-bearing molecules challenging in our L2MS experiment. The dominance of small aromatic hydrocarbons (≤14 carbons) in the LDI-MS and L2MS of the extracted soluble and insoluble OM and of the bulk rock is consistent with the low thermal maturity of the sediment and contrasts with the predominance of larger polycyclic aromatic structures commonly observed in meteorites with these techniques. We detected inorganic ions, in particular VO+, in demineralized OM that likely originate from geoporphyrins, which derive from chlorophylls during sediment diagenesis. Finally, insoluble OM yielded distinct compositions compared with extracted soluble OM, with a greater abundance of ions of mass-to-charge ratio (m/z) over 175 and additional N-moieties. This highlights the potential of laser-assisted MS to decipher the composition of macromolecular OM, in particular to investigate the preservation of biomacromolecules in microfossils. Studies comparing diverse biogenic and abiogenic OM are needed to further assess the use of this technique to search for biosignatures.

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

Toward Comprehensive Analysis of the 3D Chemistry of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are structured communities consisting of cells enmeshed in a self-generated extracellular matrix usually attached to a surface. They contain diverse classes of molecules including polysaccharides, lipids, proteins, nucleic acids, and diverse small organic molecules (primary and secondary metabolites) which are organized to optimize survival and facilitate dispersal to new colonization sites. In situ characterization of the chemical composition and structure of bacterial biofilms is necessary to fully understand their development on surfaces relevant to biofouling in health, industry, and the environment. Biofilm development has been extensively studied using confocal microscopy using targeted fluorescent labels providing important insights into the architecture of biofilms. Recently, cryopreparation has been used to undertake targeted in situ chemical characterization using Orbitrap secondary ion mass spectrometry (OrbiSIMS), providing a label-free method for imaging biofilms in their native state. Although the high mass resolution of OrbiSIMS enables more confident peak assignments, it is still very challenging to assign most of the peaks in the spectra due to complexity of SIMS spectra and lack of automatic peak assignment methods. Here, we analyze the same OrbiSIMS depth profile data generated from the frozen-hydrated biofilm, but employ a new untargeted chemical filtering process utilizing mass spectral databases to assign secondary ions to decipher the large number of fragments present in the SIMS spectra. To move towards comprehensive analysis of different chemistries in the sample, we apply a molecular formula prediction approach which putatively assigns 81% of peaks in the 3D OrbiSIMS depth profile analysis. This enables us to catalog over 1000 lipids and their fragments, 3500 protein fragments, 71 quorum sensing-related molecules (2-alkyl-4-quinolones and N-acylhomoserine lactones), 150 polysaccharide fragments, and glycolipids simultaneously from one data set and map these separated molecular classes spatially through a Pseudomonas aeruginosa biofilm. Assignment of different chemistries in this sample facilitates identification of differences between biofilms grown on biofilm-promoting and biofilm-resistant polymers.

Cell and tissue imaging by secondary ion mass spectrometry.

This Tutorial focuses on the use of secondary ion mass spectrometry for the analysis of cellular and tissue samples. The Tutorial aims to cover the considerations in sample preparation analytical set up and some specific aspects of data interpretation associated with such analysis.

Label-Free Imaging of Solid-Phase Peptide Synthesis Products and Their Modifications Tethered in Microspots Using Time-of-Flight Secondary Ion Mass Spectrometry.

Time-of-flight secondary ion mass spectrometry is used to analyze solid-phase synthesis products in 60 µm spots of high-density peptide arrays. As a result, a table of specific fragments for the individual detection of amino acids and their side chain protecting groups within peptides is compiled. The specific signal of an amino acid increases linearly as its number increases in the immobilized peptide. Mass-to-charge ratio values are identified that can distinguish between isomers such as leucine and isoleucine. The accessibility of the N-terminus of polyalanine will be studied depending on the number of its residues. The examples provided in the study demonstrate the significant potential of time-of-flight secondary ion mass spectrometry for high-throughput screening of functional groups and their accessibility to chemical reactions occurring simultaneously in hundreds of thousands of microreactors on a single microscope slide.

Nanoprojectile Secondary Ion Mass Spectrometry Enables Multiplexed Analysis of Individual Hepatic Extracellular Vesicles.

Extracellular vesicles (EVs) are nanoscale lipid bilayer particles secreted by cells. EVs may carry markers of the tissue of origin and its disease state, which makes them incredibly promising for disease diagnosis and surveillance. While the armamentarium of EV analysis technologies is rapidly expanding, there remains a strong need for multiparametric analysis with single EV resolution. Nanoprojectile (NP) secondary ion mass spectrometry (NP-SIMS) relies on bombarding a substrate of interest with individual gold NPs resolved in time and space. Each projectile creates an impact crater of 10-20 nm in diameter while molecules emitted from each impact are mass analyzed and recorded as individual mass spectra. We demonstrate the utility of NP-SIMS for statistical analysis of single EVs derived from normal liver cells (hepatocytes) and liver cancer cells. EVs were captured on antibody (Ab)-functionalized gold substrate and then labeled with Abs carrying lanthanide (Ln) MS tags (Ab@Ln). These tags targeted four markers selected for identifying all EVs, and specific to hepatocytes or liver cancer. NP-SIMS was used to detect Ab@Ln-tags colocalized on the same EV and to construct scatter plots of surface marker expression for thousands of EVs with the capability of categorizing individual EVs. Additionally, NP-SIMS revealed information about the chemical nanoenvironment where targeted moieties colocalized. Our approach allowed analysis of population heterogeneity with single EV resolution and distinguishing between hepatocyte and liver cancer EVs based on surface marker expression. NP-SIMS holds considerable promise for multiplexed analysis of single EVs and may become a valuable tool for identifying and validating EV biomarkers of cancer and other diseases.