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. 2025 Dec 27;42(1):313–321. doi: 10.1021/acs.langmuir.5c03977

An Optimized Positive Staining Protocol Method for Clear Visualization of Extracellular Vesicles by TEM Using Uranyless and Lead Citrate

Frederic St-Denis-Bissonnette †,‡, Barry Ngo †,‡, Hala Halabi †,‡, Anna Korobkow †,‡, Jianqun Wang §, Lisheng Wang ‡,∥,*, Jessie R Lavoie †,‡,*
PMCID: PMC12810375  PMID: 41454874

Abstract

Transmission electron microscopy (TEM) is widely used for visualizing extracellular vesicles (EVs) due to its nanometer-scale resolution. However, existing sample preparation protocols often lack sufficient methodological detail, requiring researchers to rely on trial-and-error approaches to achieve high-quality images. Here, we report an optimized positive-staining method that utilizes the sequential application of Uranyless and lead citrate with an intermediate buffered wash. This approach yields high image contrast, preserves EV morphology, and significantly reduces background artifacts. Staining specificity and elemental localization were confirmed using Energy-Dispersive X-ray Spectroscopy (EDS). The protocol is reproducible and robust, offering a reliable framework for EV structural analysis and potentially serving as a reference standard for TEM-based EV imaging.

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Introduction

Extracellular vesicles (EVs) are small, lipid bilayer-enclosed particles secreted by virtually all cell types. They play pivotal roles in intercellular communication by transporting a variety of bioactive molecules, including proteins, lipids, and nucleic acids. EVs have garnered significant attention in recent years due to their involvement in various physiological and pathological processes, including immunomodulation, tumor progression, the dissemination of infectious agents, and their emerging potential in diagnostic and therapeutic applications. −

A detailed understanding of EV morphology and size distribution is essential for their characterization. Among the available imaging techniques, transmission electron microscopy (TEM) stands out as the gold standard for its ability to provide high-resolution images of EVs. , It is among the techniques recommended in the MISEV2023 guidelines and is increasingly adopted in recent EV research publications. However, visualizing EVs by TEM remains challenging due to the inherently low electron density of their lipid and protein components, as well as their structural vulnerability to staining and dehydration artifacts introduced during sample preparation and imaging. , Furthermore, most published protocols do not provide sufficient details to enable reproducibility in independent studies.

Different staining methods are essential for enhancing image contrast and compensating for the inherently low electron density of carbon-rich biological materials such as EVs. Positive and negative staining are two techniques used in TEM to enhance visualization. Positive staining involves the direct binding of heavy metal salts (e.g., uranyl acetate, Uranyless, lead citrate) to cellular structures, thereby enhancing electron density and internal contrast. Conversely, negative staining enhances visualization by staining the background rather than the particles themselves, preserving external morphology but offering limited resolution for internal structures. Specialized staining techniques, such as immunogold labeling, can enable the detection of specific surface markers important for diagnosis and therapeutic applications. For example, our group previously demonstrated the successful detection of spike protein on the surface of spike-engineered EVs using immunogold-labeling.

Traditional positive staining methods rely on uranyl acetate, which provides excellent contrast but carries significant safety concerns due to its radioactive and toxic nature. Safer alternatives, such as Uranyless (nonradioactive), offer comparable contrasting capabilities and have become the standard contrast agent for positive staining in TEM imaging. The combination of Uranyless and lead citrate offers a robust approach for enhancing electron contrast in biological samples. Uranyless preferentially stains lipid membranes, while lead citrate binds to negatively charged macromolecules, further improving structural detail. Despite their complementary staining properties, the combined application of Uranyless and lead citrate for EV visualization remains largely underexplored, highlighting the need for optimized protocols to improve image quality and preserve EV structural morphology.

To validate the presence and distribution of staining elements at the nanoscale, energy-dispersive X-ray Spectroscopy (EDS) can be used alongside TEM as a complementary analytical technique. EDS enables nanoscale elemental analysis, providing critical confirmation of stain localization and composition within the sample. When a sample is exposed to an electron beam during TEM imaging, it emits characteristic X-rays that can be detected and analyzed by EDS to identify the presence of specific elements. , EDS measures the energy and intensity of X-rays emitted from the sample, where the energy is element-specific, and intensity is related to the element’s abundance. In the context of EV imaging, EDS is beneficial for confirming the presence of heavy metal stains such as Uranyless (containing lanthanum and gadolinium), lead citrate and gold (when performing immunogold labeling). Mapping of these heavy metals enables researchers to correlate their presence with regions of elevated, oxygen, and phosphorus content, indicative of biological materials such as EV membranes. This correlation helps confirm that the observed structures represent genuine biological materials rather than nonspecific background artifacts. The background area represents the grid material with little carbon presence. As such, EDS provides additional confidence that observed structures have been properly stained, thereby validating the imaging quality and interpretation. This is especially important when optimizing staining protocols for small, low-contrast biological structures, such as EVs, where distinguishing vesicles from background artifacts can be challenging. Additionally, EDS can help researchers visualize where precipitates or background artifacts form and assess their contribution to the overall background noise. Therefore, it enables researchers to optimize washing steps, adjust stain concentrations, and even select alternative stains that minimize such artifacts, thereby directly improving image quality and reducing background noise.

This study aims to systematically evaluate staining protocols and establish an optimized, reproducible method for high-resolution imaging of EVs using TEM and EDS, leveraging the complementary staining properties of Uranyless and lead citrate. Natural killer cell-derived EVs (NK-EVs) were produced and characterized as previously described. ,, As shown in these publications, NK-EVs ranged from 71.48–161.34 nm, according to NTA. The bioreactor-based origin and isolation method (99.91% purity) markedly reduces non-EV particles (NEVP) contamination, as assessed by TEM. This is contextually relevant, as most EV preparations do not achieve this level of purity, especially those derived from blood or plasma, where NEVP (e.g., lipoproteins) are often misidentified as EV particles. Our work advances existing EV research by providing a detailed and reproducible protocol for TEM-based analysis of EV structure and heterogeneity, offering more accurate and in-depth characterization of these nanoscale biological entities.

Experimental Section

Transmission Electron Microscopy (TEM) Analysis of a Single EV

Purified NK-EV samples (3 × 109 EVs/mL) were added to a Vivaspin 300 kDa filter (Sartorius, cat#VS0651), pre-equilibrated with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, cat#15710) and centrifuged at 2000g for 3 min. NK-EVs were produced and isolated as previously described. , Next, the NK-EVs, collected in the filter (not the flow-through), were washed three times with 100 μL of 4% PFA to ensure complete collection. The resulting wash volumes were combined into a low-bind Eppendorf tube to yield 300 μL of fixed-EV solution. EVs were incubated in PFA for at least 24 h at 4 °C before further processing. Three technicians independently tested each staining procedure. Later, a 25 μL drop of fixed-EV solution was added to parafilm, where an argon glow-discharged (45 s) Formvar-carbon coated EM grid (Electron Microscopy Sciences; cat#FCF300-CU) was added on top of the fixed-EV drop and rested for 10 min. Glow discharging renders the TEM grid surface hydrophilic, promoting even adsorption of aqueous EV suspensions. Next, each grid was rinsed in one drop of 50 μL filtered D-pbs–/– for 3 min before processing for contrast staining. After each incubation step, the edge of the grid was gently blotted on absorbent paper to remove excess fluid. Grids were placed upside-down (glow-discharged side down) in various combinations of UranyLess EM Stain, containing lanthanum and gadolinium, (Electron Microscopy Sciences; cat#22409) and lead citrate (Electron Microscopy Sciences; cat#22410) as follows – see Figure : Protocol A (Uranyless only): Uranyless for 30 s. Protocol B (lead citrate only): lead citrate for 30 s. Protocol C (Concurrent Uranyless + lead citrate): 1:1 mixture of Uranyless and lead citrate for 30 s. Protocol D (Sequential Uranyless + lead citrate): Uranyless for 30 s, then in lead citrate for 30 s (no in-between PBS wash). Protocol E (Sequential lead citrate + Uranyless): lead citrate for 30 s, then in Uranyless for 30 s (no in-between PBS wash). Protocol F (Sequential Uranyless + PBS + lead citrate): Uranyless for 30 s, then PBS for 30 s, then lead citrate for 30 s. Protocol G (Sequential lead citrate + PBS + Uranyless): lead citrate for 30 s, then PBS for 3 s, then Uranyless for 30 s. One unstained grid was used as a negative control. All grids were washed for 3 min with PBS, five times immediately after contrast staining. Lastly, grids were air-dried for at least 30 min before imaging on the FEI Tecnai G2 F20 field emission TEM, operating at 120 kV, at the Carleton Nano Imaging Facility, Canada. Air drying ensures complete solvent evaporation from the grid, preventing sample drift or beam-induced artifacts during TEM imaging. The images were acquired with a Gatan ORIUS TEM CCD Camera with a 4 M (2k pixels x 2k pixels) resolution at 200 and 500 nm scales. The X-ray spectra of images were collected with an X-Max 80 mm2 Energy Dispersive X-ray Spectroscopy (EDS) detector (Oxford Instruments, Abingdon, UK). EDS collection was performed in TEM mode, using a parallel electron beam focused on a defined area for several seconds or minutes to acquire the spectrum. The beam was converged to fit within each designated detection area, which was smaller than the diameter of the EV particle, as outlined by a red circle in each TEM image (see example in Figure S1). The acquisition time ranged from 40 to 60 s, with a detector dead time below 30%. EDS spectra were used to evaluate EV contrast and background staining by quantifying the presence and distribution of key staining elements (lead, lanthanum, gadolinium) relative to biologically rich regions. All steps were performed at room temperature.

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Schematic of the transmission electron microscopy (TEM) sample preparation workflow. Step 1: Grids are glow-discharged for 45 s to increase surface hydrophilicity. Step 2: A 25 μL drop of fixed EV suspension (in 4% paraformaldehyde) is placed on parafilm, and the grid is laid on top for 10 min to allow adsorption. Grids are then washed in D-pbs for 3 min. Step 3: Positive staining is applied to enhance image contrast. Protocols A–G represent different combinations and sequences of Uranyless and lead citrate, with or without PBS rinses. The edge of each grid is gently blotted on absorbent paper between steps. Step 4: Grids are air-dried for at least 30 min before TEM imaging and EDS sampling. Step 5: Images and elemental data are analyzed using TEM and EDS.

EV-TRACK

We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV250068).

Statistical Analysis

Data were expressed as the means ± the standard deviation (S.D.) or the standard error of the mean (S.E.M.). The data were normalized to the control group for relative comparison, as indicated in each figure legend. The number of experimental and technical replicates used is indicated in the figure legends. Statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software Inc., LaJolla, CA, USA), where a p-value of <0.05 was considered statistically significant and significance differences are marked with a single (p < 0.05), double (p < 0.01), triple (p < 0.001), or quadruple (<0.0001) asterisk. Two-way ANOVA followed by post hoc tests (Tukey’s or Sidak’s multiple comparisons) or the Kruskal–Wallis test followed by Dunn’s post hoc test is used, as indicated in the figure legend. The EV surface areas were measured using the Fiji software, and their diameters were calculated using the formula d=4*Aπ , where d is the EV diameter and A is the measured EV surface area. Signal-to-noise ratios (SNRs) were calculated from EDS spectra by comparing stain-specific elemental counts (lanthanum, gadolinium, and/or lead) in EV-rich regions versus adjacent background areas on the same TEM grid. EV regions were identified by the carbon signal. SNR was calculated as SNR=MeanElementalIntensityEVregionMeanElementalIntensityBackgroundregion . Values were averaged across ≥3 replicates per protocol. Higher SNRs reflect greater stain specificity and lower background deposition.

Results and Discussion

Comparison of Multiple Staining Protocols

To optimize the positive staining protocol for EVs, multiple TEM staining protocols were tested, including various combinations and sequences of Uranyless and lead citrate application, to assess image contrast, EV structural integrity, and background clarity The protocols, as shown in the experimental workflow (Figure ), included key steps such as glow discharge, EV adsorption onto grids, staining, and drying. For each protocol, approximately 10–15 TEM images were acquired at a scale of 200 and 500 nm per technical replicate. A total of 45 vesicles (15 per technical replicate across 3 technical replicates, each performed by a different user) were analyzed per protocol to determine the average EV surface area and diameter (Figure ). Additionally, Energy-dispersive X-ray spectroscopy (EDS) measurements of EVs and background were performed on both EVs and background regions, with 2–4 spectra collected at each scale (200 and 500 nm TEM images) per technical replicate (Figure ). Representative EDS measurements are shown in Figure S1.

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Comparison of positive-staining protocols for visualizing NK-EVs using TEM. A) Representative TEM images of NK-EVs stained with various combinations of Uranyless (UL) and lead citrate (LC) as per Protocols A to G, compared to unstained NK-EVs (scale bars: top row = 200 nm, bottom row = 500 nm). B) Quantification of NK-EV diameters (nm) across different staining protocols. C) Quantification of EV surface area (nm) across different staining protocols. Data are represented as mean ± SEM from 45 single vesicles (15 vesicles x 3 replicates). Statistical analysis was performed using Kruskal–Wallis One-way ANOVA followed by posthoc Dunn’s multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****), ns = not significant.

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Elemental analysis of stained NK-EVs and background using Energy-Dispersive X-ray Spectroscopy (EDS). A) Background EDS spectra across all staining protocols (Protocols A to G), showing the elemental composition of grid regions lacking NK-EVs. B) EDS spectra of NK-EV-rich regions for each staining condition. C) Quantitative comparison of elemental abundance: i) Abundance of key staining agents (lanthanum, gadolinium, lead) within NK-EV-containing regions; ii) Corresponding abundance in background areas; iii) Signal-to-noise ratios were calculated as the abundance of staining elements in NK-EV-rich regions versus background. Data are shown as mean ± SEM from ≥5 replicates. The legend for all elements analyzed is shown.

Image Quality Across the Different Protocols

Protocol A (Uranyless-only) exhibited high image contrast and minimal background noise (Figure ). However, the EVs were somewhat deformed, with an average diameter of around 76 nm and reduced membrane definition. EDS results showed a minimal presence of lanthanum and gadolinium (components found in Uranyless) in the background, with a relatively high signal-to-noise ratio (SNR) (Figure ). Conversely, protocol B (lead citrate-only) demonstrated high image contrast and very minimal background noise (Figure ). The EDS results showed a minimal presence of lead found in the lead citrate compound in the background, with a relatively low SNR (Figure ). However, the EVs were significantly deformed, with an average diameter of around 45 nm. Protocols involving a single agent staining step, such as Protocols A and B, often resulted in suboptimal imaging due to excessive background noise, as demonstrated in Protocol A or to inadequate electron density, as demonstrated in Protocol B.

Protocol C (Concurrent Uranyless + lead citrate) demonstrated high image contrast and some EV structural integrity (Figure ). However, the background noise was poor as notable salt crystal formation surrounded the EVs, and the EDS showed a significant amount of lanthanum and gadolinium in the background with low-moderate SNR for both staining agents (Figure ). Protocol D (Sequential Uranyless + lead citrate) demonstrated high image contrast but low EV structural integrity, with the average diameter ranging between 30 and 70 nm across replicates (Figure ). The background noise was moderately high due to some salt precipitation surrounding the EVs, and the EDS revealed the presence of sodium and hydroxide ions in the background, resulting in a low-moderate SNR (Figure ). Protocol E (Sequential lead citrate + Uranyless) demonstrated high image contrast and some EV structural integrity (Figure ). However, the amount of EVs on the grid was markedly reduced, and the results were not easily reproducible. Background noise and EDS results showed a low SNR for the UL stain but a high SNR for the LC stain (Figure ). Protocol F (Sequential Uranyless + PBS wash + lead citrate) demonstrated high image contrast, minimal EV deformation, and minimal background noise (Figure ). The EDS results showed virtually no lead and lanthanum and minimal sodium and oxygen in the background, thereby exhibiting the highest SNR for LC stain, indicating optimal stain specificity and minimal nonspecific deposition (Figure ). Protocol G (Sequential lead citrate + PBS wash + Uranyless) exhibited high image contrast and variable EV structural integrity (Figure ). However, some background noise was present, and the EDS results showed some lead, lanthanum, and gadolinium in the background; moderate SNR (Figure ). Although Protocol G yielded high contrast, EV morphology was partially compromised, with more salt precipitation compared to Protocol F.

Sizing assessment revealed the EV diameters across different protocols ranged from 20 to 90 nm. Notably, EV diameters in Protocols B, C, D, and G were smaller compared to Protocols A, E, and F. This discrepancy is not due to sample heterogeneity (same starting material) but to a methodological artifact. Protocols B and G yielded the smallest EVs, potentially due to the alkaline environment (pH ∼ 12) provided by lead citrate stain that might affect the conformation of proteins on the EV surface or affect the lipid bilayer integrity. , Uranyless, on the other hand, has a more neutral pH, which may exert less structural stress on EVs and help preserve their morphology closer to the native state. In addition, Uranyless, following a PBS wash, might help buffer the high alkalinity of lead citrate, thereby contributing to the preservation of EV structural integrity, as observed in Protocol F. Notably, the size distribution determined in Protocol F closely aligns with NTA measurement by NanoSight (71.48–161.34 nm7), suggesting that this method best preserves the vesicles’ morphology. Based on these findings, we recommend that studies reporting EV morphology include a detailed, step-by-step description of their staining protocol, specifying the order of stain application, the use of intermediate washes, and stain incubation times, to enhance reproducibility and ensure accurate structural interpretation.

Altogether, these findings support the importance of stain sequence and intermediate washing to optimize EV visualization (Figures -). Table summarizes these findings. This systematic comparison of staining protocols reveals that the choice and sequence of staining agents have a profound and direct impact on the EV morphology, image quality and the interpretation thereof. This finding aligns with the broader principle that significant variability in EV morphology and size is protocol-dependent. For instance, negative staining methods involving uranyl acetate or Uranyless often resulted in cup-shaped EV morphology. − However, our positive staining technique did not appear to yield a visible cup-shaped morphology in EVs, possibly due to the stain deposition obscuring the curvature of the cup shape. A recent comparative study of uranyl-free stains further underscores the need for continued investigations into how staining protocols influence the visualization of EV ultrastructure.

1. Summary of Staining Protocol Performance for TEM Visualization of NK-EVs. Legend: UL: Uranyless; LC: Lead Citrate, SNR: Signal-To-noise Ratio.

  Staining Sequence EV Morphology Image Contrast Background Noise/Artifacts EDS Confirmation (SNR of UL) EDS Confirmation (SNR of LC) Reproducibility Overall Performance
A Uranyless only Mild deformation, fuzzy edges High (++ +) Minimal High (∼11) Not applicable High (3/3) Acceptable
B Lead citrate only Significant deformation Moderate (++) Minimal Not applicable Low (∼6) High (3/3) Suboptimal
C UL + LC (concurrent mix) Some preservation High (++ +) High salt precipitation Low–Moderate (∼3) Low–Moderate (∼11) Moderate (2/3) Suboptimal
D UL → LC (no PBS rinse) Variable; many deformed High (++ +) Moderate precipitation Moderate (∼5) Low (∼6) Moderate (2/3) Acceptable but inconsistent
E LC → UL (no PBS rinse) Some preservation; low EV yield Moderate (++) Moderate noise Low (∼1) High (∼71) Low (1/3) Not recommended
F UL → PBS → LC Well-preserved Very high (++ + +) Minimal (optimal) Low (∼0.5) High (∼70) High (3/3) Optimal (recommended)
G LC → PBS → UL Some deformation High (++ +) Moderate salt artifacts Moderate-High (∼7) Low (∼8) High (3/3) Acceptable
x No stain Native morphology? Very low (−) None Not applicable Not applicable High Baseline (comparison only)
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It is also important to note that image quality is influenced not only by the staining protocol, but also by EV purity and the source material. For instance, conditioned media generally contain fewer non-EV particles (NEVP) than serum/plasma, which are rich in lipoproteins that can resemble EVs under TEM. In this study, the NK-EVs used were previously shown to have exceptionally high purity (99.91%), resulting in minimal visible contaminants. This significantly reduced the likelihood of background artifacts and misidentification of lipoproteins as EVs. , It remains to be determined if Protocol F can maximize image quality (i.e., SNR and EV versus NEVP identification) with samples of lesser purity (i.e., EV purified from serum or plasma), just as well as demonstrated here.

Lastly, EDS plays a critical role in confirming effective EV staining by mapping elements from the staining agents to the organic components of EVs, as well as in evaluating the potential background artifacts introduced by the staining process. To date, no studies have systematically applied EDS to assess the distribution of staining elements within both EVs and the surrounding background. While EDS has been applied in some studies, it has primarily been used on unstained samples to determine the mineral composition of EVs. − Conversely, Bose et al. 2018, utilized EDS to confirm the presence of uranyl acetate in engineered EVs and to analyze the elemental composition; however, their study did not include the background staining. These observations, including ours, underscore the importance of EDS in validating image quality across various staining protocols.

Protocol F - Leading Method for High-Contrast EV Visualization

Comparatively, the most contrast-efficient staining protocol was identified as Protocol F (Sequential Uranyless + PBS wash + lead citrate), which exhibited optimal contrast, minimal EV deformation, and reduced background staining artifacts. Quantitative evaluation of staining efficiency using EDS revealed inferior results for protocols A, B and D. Conversely, protocols E or C showed poor reproducibility or excessive background staining. Across three replicates, Protocol F demonstrated consistent results, with minimal background noise and EV deformation and maximal EV staining contrast. Specific artifacts, such as salt precipitations, were observed in Protocol C but were absent in Protocol F. EV deformations were observed in Protocols B and D but were absent in Protocols C, F, and G. Detailed EDS assessment of Protocol F revealed carbon, oxygen, phosphorus accounted for 86.7 ± 3.7%, 1.6 ± 0.5% and 0.3 ± 0.1 of the background area, but 37.9 ± 8.7%, 12.0 ± 2.3% and 2.8 ± 0.6% of the NK-EV area (Figure ). These distributions are consistent with the data shown in Figure and Figure S1. The colocalization of staining elements corresponding to carbon/oxygen/phosphorus-rich EVs supports the selective membrane staining while reducing the likelihood of misinterpreting background artifacts as vesicle structures.

The sequential application of Uranyless/lead citrate (Protocol F) leverages the high electron density properties of Uranyless and the binding properties of lead citrate, allowing for enhanced EV visualization without excessively compromising their native morphology. The PBS rinses between staining steps were crucial in reducing nonspecific binding and background artifacts, as observed in the control experiments. This step washes away excess, unbound heavy metal ions from the initial Uranyless staining, thereby minimizing salt precipitation when lead citrate is applied. Without PBS wash (Protocol D), a higher incidence of salt precipitation and background noise was observed. Additionally, while combined Uranyless and lead citrate (Protocol C) potentially generated high contrast, it exhibited a significantly higher incidence of salt precipitation, suggesting the need for the sequential application of Uranyless and lead citrate, followed by a PBS intermediate wash to achieve a clean, high-quality image. Notably, the sequence of Uranyless and lead citrate application is also crucial, as Uranyless serves as a primary stain while lead citrate functions as a contrast enhancer.

Taken together, Protocol F (sequential Uranyless and lead citrate staining, with an intermediate PBS rinse) represents an optimized staining method that enhances the reproducibility of EV morphological assessment and may serve as a reference protocol for laboratories using TEM in EV research. This protocol is the most contrast-enhancing method for TEM-based imaging of EVs, maintaining EV structural integrity and minimizing background artifacts. The sequential application of Uranyless and lead citrate combines their complementary strengths: enhancing image contrast while preserving EV structural integrity, which minimizes artifacts and background noise. Providing a standardized and reliable staining protocol can facilitate reproducibility across laboratories and support more accurate structural analyses, ultimately accelerating progress in EV research.

Supplementary Material

la5c03977_si_001.pdf (427.6KB, pdf)

Acknowledgments

The authors thank Drs. Roger Tam, Huixin Lu and Simon Sauvé for their critical manuscript review.

Glossary

Abbreviations

EVs

Extracellular Vesicles

EDS

Energy-Dispersive X-ray Spectroscopy

LC

Lead Citrate

NK-EVs

Natural Killer Cells – derived Extracellular Vesicles

PBS

Phosphate-Buffered Saline

PFA

Paraformaldehyde

TEM

Transmission Electron Microscopy

UL

UranyLess.

The Supporting Information is available. Figure S1 demonstrates an example of sample collection by Energy-Dispersive X-ray Spectroscopy (EDS) under TEM mode. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.5c03977.

Frederic St-Denis-Bissonnette, Lisheng Wang and Jessie R. Lavoie conceptualized the project. Frederic St-Denis-Bissonnette designed the experiments. Frederic St-Denis-Bissonnette generated the NK-EV material. Barry Ngo, Hala Halabi, Anna Korobkow and Jianqun Wang performed the TEM experiments. Frederic St-Denis-Bissonnette, Barry Ngo, Hala Halabi, and Anna Korobkow performed data visualization. Frederic St-Denis-Bissonnette, Lisheng Wang and Jessie R. Lavoie managed the project. Frederic St-Denis-Bissonnette, Barry Ngo, Hala Halabi, and A. Korobkow, L. Wang and J.R. Lavoie wrote the manuscript. All authors read and approved the manuscript.

This work was supported by operating grants from the Genomics Research and Development Initiative (GRDI) Phase VII–VIII (2019–2030) from the Government of Canada obtained by JRL and LW, as well as operating grants from the Natural Sciences and Engineering Research Council RGPIN-2019–05220, Cancer Research Society 24064, the Canadian Institutes of Health Research (CIHR) Operating Grant 175177 obtained by LW.

The authors declare no competing financial interest.

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