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Imaging Single Virus Particles on the Surface of Cell Membranes by High-Resolution Scanning Surface Confocal Microscopy

Andrew I. Shevchuk ^*, Phil Hobson , Max J. Lab ^*, David Klenerman , Nina Krauzewicz  and Yuri E. Korchev ^*

^* Division of Medicine and MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom; and Department of Chemistry, Cambridge University, Cambridge CB2 1EW, United Kingdom

Correspondence: Address reprint requests to Y. Korchev, Division of Medicine, Imperial College London, MRC Clinical Sciences Centre, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Tel.: 44-208-383-3080; Fax: 44-208-383-8306; E-mail: y.korchev{at}ic.ac.uk.

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.