Subnanometre single-molecule localization, registration and distance measurements

Seeing single molecules It is possible to bypass the diffraction limit that restricts the resolution of an optical microscope to about half the wavelength of the incident light by using charge-coupled devices (CCDs) to track fluorescent probe molecules attached to biological molecules of interest. This strategy lowers the limit for imaging single molecules or intramolecular distances from a diffraction-limited 200 nanometres to nearer 20 nanometres. Now Steven Chu and colleagues use a modified form of this CCD-fluorescence technology to resolve distances with subnanometre precision in an otherwise conventional far-field fluorescence imaging system. They use a feedback system to compensate for the non-uniform response of the CCD silicon array to incoming photons — which may be an artefact of chip manufacture — that, unchecked, blurs the pixels representing points within a few nanometres of one another. This resolution should allow the characterization of the components of large, multi-protein biological complexes. The method should inspire similar improvements in nanotechnology or astronomical measurements that also rely on digital cameras.