Abstract
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. Researchers have demonstrated a super-resolution imaging technique that allows the direct observation of adenoviruses of 75 nm in size without staining or the use of fluorescent labeling. The approach, reported by Lin Li and co-workers at the University of Manchester, UK, achieves imaging well beyond the diffraction limit by employing glass microspheres submerged in water. A 100-μm-diameter BaTiO3 microsphere is placed on the object being imaged, illuminated with white light. The microsphere captures near-field evanescent waves and converts them via frustrated total internal reflection into propagating far-field waves, which can be imaged by a conventional optical microscope. The researchers also used the method to image 100-nm-spaced periodic structure of a DVD Blu-Ray disk and 50 nm nanopores in a sample of anodic aluminium oxide.
Highlights
Optical microscopic imaging resolution has a theoretical limit of approximately 200 nm within the visible light spectrum due to the far-field diffraction limit, which prevents the technique from being used for direct observation of live viruses
We report the use of submerged microsphere optical nanoscopy (SMON) for the direct imaging of an adenovirus with a diameter of 75 nm at a resolution beyond the optical diffraction limit
Demonstration of super-resolution imaging by SMON To demonstrate the super-resolution capability of large-microsphere (100 mm) SMON and to calibrate the imaging system, a Blu-Ray DVD disk with approximately 100-nm line spacing was imaged using an optical microscope (503, NA50.75) with a 100-mm diameter BaTiO3 microsphere in water under white light illumination
Summary
Optical microscopic imaging resolution has a theoretical limit of approximately 200 nm within the visible light spectrum due to the far-field diffraction limit, which prevents the technique from being used for direct observation of live viruses (typically 5–150 nm, with some up to 300 nm). Fluorescence optical microscopy is a recently established method for the imaging of cellular structures, bacteria and viruses beyond the optical diffraction limit, down to a resolution of 6 nm.[1,2,3,4] This technique is based on the detection of light emitted by the fluorescing specimen when it is excited by light of a specific wavelength. There has been no report demonstrating white light direct optical imaging of viruses below 100 nm in size
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
