title: An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles.
abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.
authors.affiliation: Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China; Department of Biotechnology, Nanchang University, Nanchang, Jiangxi 330031, China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China; Department of Biotechnology, Nanchang University, Nanchang, Jiangxi 330031, China. Electronic address: dr_yongchen@hotmail.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000388:Air; D055252:Cell-Derived Microparticles / Q000502:physiology* / Q000648:ultrastructure*; D003890:Desiccation / Q000379:methods; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D018613:Microscopy, Confocal; D036103:Nanotechnology / Q000379:methods
keywords: Atomic force microscopy (AFM); Endothelial cells; Membrane vesicles; Vesicle-derived pits