Although challenging, single-virus imaging has revealed key steps in the viral life cycle, and it will likely reveal more about viruses as imaging equipment and techniques improve. In a review, researchers from Harvard University in Cambridge, Mass., have explained the benefits, challenges and future of tracking single viruses.
The reviewers emphasized the advantages of using cellular structures tagged with fluorescent proteins in combination with viruses labeled with fluorophores. The combination can enable observation of even brief interactions between viruses and cellular proteins, including mechanisms in which viruses enter cells and the way that they disassemble upon entry. Dual labeling of cellular structures and viruses also can reveal how viruses travel through and exit the cell.
The reviewers noted some of the challenges of imaging single viruses and suggested solutions. For example, viruses are small, so fluorophores may become crowded on a single virus. Because crowded fluorophores may self-quench, it is important to use a number of fluorophores consistent with the size of the particular virus. Cells can autofluoresce, so it is better to use fluorophores that absorb at longer wavelengths. Fluorescent tags may interfere with viral form and function, so potential negative effects of the fluorescence probes on the viruses should be examined, and alternative labeling methods should be considered. Live-cell imaging produces gigabytes of data, but recent advancements have permitted the analysis and assembly of images into a comprehensive depiction of the viral life cycle.
The reviewers suggested several strategies to improve single-virus imaging. They called for optical methods that can reach the nanometer scale, brighter and smaller probes, high-throughput screening with greater resolution, and in vivo methods such as nonlinear optical microscopy and endoscopy. (Nature Reviews Microbiology, March 2007, pp. 197-208.)
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