An innovative approach to calibrating high-tech microscopes enables researchers to track the movement of single molecules in 3D at the nanoscale.
A Stanford University research team, led by W. E. Moerner, extends the work that earned Moerner and colleagues Eric Betzig and Stefan W. Hell the 2014 Nobel Prize for Chemistry. Betzig and Moerner pioneered the development of super-resolution imaging, which broke the diffraction limit of optical microscopy by using the fluorescence of single molecules for the first time. The new work, published in The Optical Society’s high impact journal Optica, demonstrates a marked improvement in the accuracy of this imaging technique and for tracking molecules in three dimensions.
Ref: Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy. Optica (2015) | DOI: 10.1364/OPTICA.2.000985
The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope’s field of view has not yet been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50–100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and superresolution methods demanding high accuracy.