SUPER-RESOLUTION MICROSCOPY/SINGLE MOLECULE IMAGING: Interactive feedback enables measurement of bio molecules to 0.5 nm-optically

As reported in the journal Nature, US Energy Secretary Steven Chu led the development of a technique that has proven able to image biological structures–and distances between them–in "biologically relevant environments" to resolutions as small as 0.5 nm.¹ The method uses standard optical microscopy equipment, and can improve many super-resolution, far-field imaging methods such as stochastic optical reconstruction microscopy (STORM), photo-activated localization microscopy (PALM), stimulated emission depletion (STED) and multiphoton microscopy.

The work has ability to revolutionize biology, particularly structural biology, says Chu. "One of the motivations for this work," he explains, "was to measure distances between proteins that form multi-domain, highly complex structures, such as the protein assembly that forms the human RNA polymerase II system, which initiates DNA transcription."

While it's true that non-optical systems such as electron microscopes can resolve objects well into the sub-nanometer scale, they are ill-suited for biological specimen study.

It is also true that detecting individual fluorescent labels attached to biological molecules of interest using charge-coupled devices (CCDs) has yielded resolutions as fine as 5 nm. But traditionally, this technology has been unable to image single molecules or distances between molecules less than 20 nm. Chu and co-authors Alexandros Pertsinidis and Yunxiang Zhang were able to use this same CCD-fluorescence approach–but with a critical difference.

Precise control

They note that 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 on just where a photon hits the silicon chip surface, the photon may be absorbed slightly differently, and may or may not generate a measurable charge. This non-uniform response to incoming photons, probably an artifact of chip manufacturing, produces a blurring of pixels that hampers resolution points within a few nanometers of one another.

The researchers' "secret sauce" is an active feedback system that allows sub-pixel precision placement of the image depicting a single fluorescent molecule anywhere on the array. This "enables us to work in a region smaller than the typical three pixel length-scale of the CCD non-uniformity," says Pertsinidis, 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 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 (they used a three-axis, closed-loop piezoelectric stage equipped with capacitive position sensors corrected to 0.01% with a digital controller) was key, as was the ability to calculate the geometric center (centroid) of the image. These factors made it possible to not only measure photo-response non-uniformity between pixels, but also to measure non-uniformity within each individual pixel.

"Knowing this non-uniformity then allows us to make corrections between the apparent position and the real position of the image's centroid," says Chu. "Since this non-uniform 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."

Pertsinidis is continuing to work with Chu and others on further development and application of this technique. In addition to the human RNA polymerase II system, he and the group are using it 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. Pertsinidis, Zhang, and another post-doc, Sang Ryul Park, are also using this technique to create 3D measurements of the molecular organization inside brain cells.

"The idea is to determine the structure and dynamics of the vesicle fusion process that releases the neurotransmitter molecules used by neurons to communicate with one another," Pertsinidis says. "Right now, we are getting in situ measurements with a resolution of about 10 nm, but we think we can push this resolution to within 2 nm."

In collaboration with Joe Gray, Berkeley Lab's Associate Director for Life Sciences, post-docs in Chu's research group are also using the technique to study the attachment of signaling molecules on the RAS protein, which has been linked to a number of cancers. This research could help explain why cancer therapies that perform well on some patients are ineffective on others.

1. A. Pertsinidis et al. Nature 466, 647-651 (2010)