Fascinating insights into sub-nm localization of molecules from Berkeley

Subnanometre single-molecule localization, registration and distance measurements

Some attention-getting work performed by Alexandros Pertsinidis, Yunxiang Zhang and Nobelist Steven Chu (also the United States' Secretary of the Department of Energy) has revealed nanoscale error mechanisms believed to be universal in CCD-based wide-field imaging systems. The work was published in the prestigious journal, Nature.

The team developed mapping and active-stabilization techniques and demonstrated that these can combine to improve the optical localization of fluorescent molecules by an order of magnitude, to a resolution of 0.5nm and an accuracy of 0.77nm. This represents a significant advance for fields such as FRET microscopy --important for observing the shape-changes of proteins, as it reveals their mechanical action and thereby illuminates the foundation of many diseases.

A key revelation in the research was that much of the microscopy system's ability to localize molecules optically was limited by high-spatial-frequency non-uniformities in the inter-pixel photoresponse of the CCD. These nanoscale errors, which appear worse at the interstices between pixels, have the effect of a finely-patterned crinkling of the pixel distribution, as if the map of pixel locations had been wadded up and then re-flattened. Since fluorescent molecules are localized to sub-diffraction precisions by calculating the center of their (Gaussian) Airy disk image as it illuminates many pixels, this non-uniformity limits the precision and accuracy of the localization.

The stabilization of the imaging setup is achieved by imaging an illuminated pinhole onto the CCD. This serves as a bright fiducial and can be localized to 0.3% of a pixel. Its position is used as feedback to stabilize the optical system against perturbation, using a high-speed piezoelectric tip/tilt mount. The locked-in stability of the system was measured to be 0.64nm over several hours. A separate feedback system based on the optical position of a selected fluorescent molecule continuously adjusts the XYZ piezoelectric nanopositioning stage carrying the test sample with respect to the optics. These continuous, sub-nanometer-precision processes place a premium on interface throughput and responsiveness.

Combining these tools, the researchers developed a method of calibrating small regions of the CCD, allowing localization of molecules to 0.5nm-- a factor of ten better than had previously been achieved. The technique can be extended to cover larger areas of the detector, up to its full extent.

Acknowledging the importance of recombinant innovation across fields, the authors note:
"...Our methodology might also prove valuable to characterize/design precision photometric imaging systems in fields such as atomic physics or astronomy... the subnanometre closed-loop control and registration afforded by our technique could become essential concepts in design of future sub-10-nm optical lithography tools and may allow new nanometrology applications."

Tantalizingly, the researchers state that in an upcoming publication, the mapping technique is extended to the intra-pixel level with approximately 1 Angstrom precision, promising another round of groundbreaking innovation and further insights into the activities of the molecular machines that underly all life.

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