Because of the small sizes of most viruses (typically 5-150 nm), standard optical microscopes, which have an optical diffraction limit of 200 nm, are not generally suitable for their direct observation. Electron microscopes usually require specimens to be placed under vacuum conditions, thus making them unsuitable for imaging live biological specimens in liquid environments. Indirect optical imaging of viruses has been made possible by the use of fluorescence optical microscopy that relies on the stimulated emission of light from the fluorescing specimens when they are excited with light of a specific wavelength, a process known as labeling or self-fluorescent emissions from certain organic materials. In this paper, we describe direct white-light optical imaging of 75-nm adenoviruses by submerged microsphere optical nanoscopy (SMON) without the use of fluorescent labeling or staining. The mechanism involved in the imaging is presented. Theoretical calculations of the imaging planes and the magnification factors have been verified by experimental results, with good agreement between theory and experiment.

We report a direct optical super-resolution imaging approach with 25 nm (∼ λ/17) lateral resolution under 408 nm wavelength illumination by combining fused silica and polystyrene microspheres with a conventional scanning laser confocal microscope (SLCM). The microsphere deposited on the target surface generates a nanoscale central lobe illuminating a sub-diffraction-limited cross-section located on the target surface. The SLCM confocal pinhole isolates the reflected light from the near-field subdiffractive cross-section and suppresses the noises from the side lobe and the far-field paraxial focal point. The structural detail of the subdiffractive cross-section is therefore captured, and the 2D target surface near the bottom of microspheres can be imaged by intensity-based point scanning.

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