A Platform To Enhance Quantitative Single Molecule Localization Microscopy

Golfetto, Ottavia; Wakefield, Devin L.; Cacao, Eliedonna E.; Avery, Kendra N.; Kenyon, Victor; Jorand, Raphaël; Tobin, Steven J.; Biswas, Sunetra; Gutierrez, Jennifer; Clinton, Ronald M.; Ma, Yuelong; Horne, David; Williams, John C.; Jovanović-Talisman, Tijana

doi:10.1021/jacs.8b04939

Cited by 25 publications

(50 citation statements)

References 64 publications

(170 reference statements)

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“…However, small labelling heterogeneity is expected even with optimized protocols. To minimize this effect, we calculated the average number of appearances for fluorescently labelled Abs using a surface assay for molecular isolation (SAMI) [17], Figure S2. This approach allowed us to directly test photophysical properties of labelled Abs using the same optical setup and imaging conditions as those used for detecting EVs.…”

Section: Resultsmentioning

confidence: 99%

“…The degree of labelling was calculated with a NanoDrop for each batch of labelled Abs. To minimize the effects of labelling heterogeneity on molecular counting, we defined the average number of detected localizations using the SAMI assay [17]. To calculate the photophysical properties, surfaces of sparsely attached reporters were prepared.…”

Section: Methodsmentioning

confidence: 99%

“…These surfaces were imaged under the same conditions as surfaces with EVs, described below. The resulting localizations were analysed to determine the average number of fluorophore appearances per molecule as described [17]. Average values of 2 appearances per molecule were determined for both cetuximab‐AF647 and anti CA19‐9 Ab‐AF647 (Figure S2).…”

Section: Methodsmentioning

confidence: 99%

“…Here, qSMLM was used to comprehensively assess EVs from both cell culture and patient plasma. We combined 1) affinity selection of EVs via EGFR or CA19‐9, 2) robust molecular counting [17], and 3) fast data analysis. Using this strategy, we quantified both the size and membrane protein content of individual EVs.…”

Section: Introductionmentioning

confidence: 99%

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Single molecule characterization of individual extracellular vesicles from pancreatic cancer

Lennon

Wakefield

Maddox

et al. 2019

J of Extracellular Vesicle

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Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. Here, we robustly assessed heterogeneous EV populations from cultured cell lines via nanoparticle tracking analysis, proteomics, transcriptomics, transmission electron microscopy, and quantitative single molecule localization microscopy (qSMLM). Using qSMLM, we quantified the size and biomarker content of individual EVs. We applied qSMLM to patient plasma samples and identified a pancreatic cancer-enriched EV population. Our goal is to advance single molecule characterization of EVs for early disease detection.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Single molecule characterization of individual extracellular vesicles from pancreatic cancer

Lennon

Wakefield

Maddox

et al. 2019

J of Extracellular Vesicle

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“…The average number of paGFP apperances was calculated as described before. 97 Protein densities were calculated from square ROIs of 10 μm 2 . These same ROIs were subsequently analyzed to investigate the effect of NTX and/or ethanol on MOR and KOR nano-organization.…”

Section: ■ Discussionmentioning

confidence: 99%

Ethanol and Naltrexone Have Distinct Effects on the Lateral Nano-organization of Mu and Kappa Opioid Receptors in the Plasma Membrane

Tobin

Wakefield

Terenius

et al. 2018

ACS Chem. Neurosci.

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The complex spatiotemporal organization of proteins and lipids in the plasma membrane is an important determinant of receptor function. Certain substances, such as ethanol, can penetrate into the hydrophobic regions of the plasma membrane. By altering protein−lipid and protein− protein interactions, these substances can modify the dynamic lateral organization and the function of plasma membrane receptors. To assess changes in plasma membrane receptor organization, we used photoactivated localization microscopy (PALM). This single molecule localization microscopy technique was employed to quantitatively characterize the effects of pharmacologically relevant concentrations of ethanol and naltrexone (an opioid receptor antagonist and medication used to treat alcohol use disorders) on the lateral nanoorganization of mu and kappa opioid receptors (MOR and KOR, respectively). Ethanol affected the lateral organization of MOR and KOR similarly: It reduced the size and occupancy of opioid receptor nanodomains and increased the fraction of opioid receptors residing outside of nanodomains. In contrast, naltrexone affected MOR and KOR lateral organization differently. It significantly increased KOR surface density, nanodomain size, and the occupancy of KOR nanodomains. However, naltrexone marginally affected these parameters for MOR. Pretreatment with naltrexone largely protected against ethanolinduced changes in MOR and KOR lateral organization. Based on these data, we propose a putative mechanism of naltrexone action that operates in addition to its canonical antagonistic effect on MOR-and KOR-mediated signaling.

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Cardiac troponin T in extracellular vesicles as a novel biomarker in human cardiovascular disease

Lennon

Saftics

Abuelreich

et al. 2022

Clinical & Translational Med

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Dear Editor,Soluble cardiac troponin T (cTnT), an indicator of myocardial injury and stress, is used in decision management for patients with cardiovascular disease (CVD). As highly sensitive assays can detect elevated concentrations of cTnT even in healthy individuals (e.g. outside of myocardial necrosis, electrocardiographic changes or angina) and cannot distinguish among disease conditions, 1,2 a comprehensive understanding of the cTnT-secretome is an unmet need.Within the secretome, cTnT is not only present as a soluble factor but may also be contained within extracellular vesicles (EVs). 3 EVs are nanoscale particles secreted by all cells, the cargoes of which can reflect the molecular composition of the cells of origin 4 and indicate disease or injury. 5 As EVs are easily sampled from plasma, 6 they are being developed as a 'liquid' biopsy reflecting the disease state from the tissue of origin. Here, we advanced a fluorescence-based super-resolution microscopy technique, quantitative single-molecule localization microscopy (qSMLM), to robustly characterize cTnT-positive EVs. Importantly, we provide the first report of cTnT-secretome across a spectrum of CVDs.EVs were purified from induced pluripotent stem cellderived cardiomyocyte cell media (CCM), representing a source of cardiomyocyte-derived EVs (Figure S1), and patient plasma (Figures S2): healthy subjects (n = 5), patients with heart failure (HF; n = 5), hypertrophic cardiomyopathy (n = 3), type 1 myocardial infarction (MI-TI, n = 5) or type 2 myocardial infarction (MI-TII; n = 5) and chronic kidney disease (CKD; n = 5). In all cases (Figures S1 and S2), EVs had intact morphology and contained canonical EV markers (tetraspanins CD9, CD63, CD81; luminal marker TSG101) with low amounts of soluble proteins. According to dot blots (Figure S2C), the CD81 content of patient EVs was highly variable, whereas combined tetraspanins had more uniform expression. Table S1 shows patient characteristics. Control patients wereThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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A Platform To Enhance Quantitative Single Molecule Localization Microscopy

Cited by 25 publications

References 64 publications

Single molecule characterization of individual extracellular vesicles from pancreatic cancer

Single molecule characterization of individual extracellular vesicles from pancreatic cancer

Ethanol and Naltrexone Have Distinct Effects on the Lateral Nano-organization of Mu and Kappa Opioid Receptors in the Plasma Membrane

Cardiac troponin T in extracellular vesicles as a novel biomarker in human cardiovascular disease

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