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Correcting a trick of the light brings molecules into view
Date:7/14/2010

can resolve objects well into the subnanometer scale, these systems operate under conditions not ideal for the study of biological samples. Detecting individual fluorescent labels attached to biological molecules of interest using charge-coupled devices (CCDs) arrays of silicon chips that convert incoming light into an electrical charge, has yielded resolutions as fine as five nanometers. However, until now this technology has been unable to image single molecules or distances between a pair of molecules much less than 20 nanometers.

Chu and his co-authors were able to use the same CCD-fluorescence technology to resolve distances with subnanometer precision and accuracy by correcting a trick of the light. The electrical charges in a CCD array are created when photons strike the silicon and dislodge electrons, with the strength of the charge being proportional to the intensity of the incident photons. However, depending upon precisely where a photon hits the surface of a silicon chip, there can be a slight difference in how the photon is absorbed and whether it generates a measurable charge. This non-uniformity in the response of the CCD silicon array to incoming photons, which is probably an artifact of the chip manufacturing process, results in a blurring of pixels that makes it difficult to resolve two points that are within a few nanometers of one another.

"We have developed an active feedback system that allows us to place the image of a single fluorescent molecule anywhere on the CCD array with sub-pixel precision, which in turn enables us to work in a region smaller than the typical three pixel length-scale of the CCD non-uniformity," says Pertsinidis, who is the lead author on the Nature paper. "With this feedback system plus the use of additional optical beams to stabilize the microscope system, we can create a calibrated region on the silicon array where the error due to non-uniformity is reduced to 0.5 nanometers. By placing the molecule
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Contact: Lynn Yarris
lcyarris@lbl.gov
510-486-5375
DOE/Lawrence Berkeley National Laboratory
Source:Eurekalert  

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