title: Imaging single virus particles on the surface of cell membranes by high-resolution scanning surface confocal microscopy.
abstract: We have developed a high-resolution scanning surface confocal microscopy technique capable of imaging single virus-like particles (VLPs) on the surfaces of cells topographically and by fluorescence. The technique combines recently published single-molecule-resolution ion-conductance microscopy that acquires topographical data with confocal microscopy providing simultaneous fluorescent imaging. In our experiments we have demonstrated that the cell membrane exhibits numerous submicrometer-sized surface structures that could be topographically confused with virus particles. However, simultaneous acquisition of confocal images allows the positions of fluorescently tagged particles to be identified. Using this technique, we have, for the first time, visualized single polyoma VLPs adsorbed onto the cell membrane. Observed VLPs had a mean width of 108 +/- 16 nm. The particles were randomly distributed across the cell membrane, and no specific interactions were seen with cell membrane structures such as microvilli. These experiments demonstrate the utility of this new microscope for imaging the interactions of nanoparticles with the cell surface to provide novel insights into the earliest interactions of viruses and other nanoparticles such as gene therapy vectors with the cell.
authors.affiliation: Division of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom.
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Shevchuk
authors.forename: Phil
authors.initials: P
authors.lastname: Hobson
authors.forename: Max J
authors.initials: MJ
authors.lastname: Lab
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.forename: Nina
authors.initials: N
authors.lastname: Krauzewicz
authors.forename: Yuri E
authors.initials: YE
authors.lastname: Korchev
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000648:ultrastructure*; D002522:Chlorocebus aethiops; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000295:instrumentation*; D012680:Sensitivity and Specificity; D014771:Virion / Q000648:ultrastructure*