Secretary of Energy Chu leads development of superresolution technique

Berkeley, CA--Steven Chu--Secretary of Energy, Nobel laureate, and former director of the Lawrence Berkeley National Laboratory--has led the development of a medical imaging technique that enables the use of optical microscopy to image objects or the distance between them at resolutions as small as 0.5 nm, an order of magnitude smaller than the previous best.¹ A DNA molecule measures about 2.5 nm in width.

"The ability to get subnanometer resolution in biologically relevant aqueous environments has the potential to revolutionize biology, particularly structural biology," says Secretary Chu. "One of the motivations for this work, for example, was to measure distances between proteins that form multidomain, highly complex structures, such as the protein assembly that forms the human RNA polymerase II system, which initiates DNA transcription."

While nonoptical imaging systems, such as electron microscopes, 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 CCDs has yielded resolutions as fine as 5 nm; however, until now, this technology has been unable to image single molecules or distances between a pair of molecules much less than 20 nm.

Correcting for a CCD nonuniformity
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 CCD peculiarity. 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 nonuniformity 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 nonuniformity," says Alexandros Pertsinidis, the lead author of the 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 nm. By placing the molecules we want to measure in the center of this region we can obtain subnanometer resolution using a conventional optical microscope that you can find in any biology lab."

Chu says that the ability to move the stage of a microscope small distances and calculate the centroid of the image makes it possible to not only measure the photoresponse nonuniformity between pixels, but also to measure the nonuniformity within each individual pixel.

"Knowing this nonuniformity then allows us to make corrections between the apparent position and the real position of the image's centroid," says Chu. "Since this nonuniform response is built into the CCD array and does not change from day to day, our active feedback system allows us to image repeatedly at the same position of the CCD array."

Other uses
In addition to imaging the human RNA polymerase II system, the group is using the technology to determine the structure of the epithelial cadherin molecules that are responsible for the cell-to-cell adhesion that holds tissue and other biological materials together.

In addition to its biological applications, the superresolution technique could also help characterize and design precision photometric imaging systems in atomic physics or astronomy, and allow for new tools in optical lithography and nanometrology.

REFERENCE:

1. Alexandros Pertsinidis et al., Nature, published online 07 July 2010, doi:10.1038/nature09163