Calibration Sharpens Superresolution Microscopes

Superresolution microscopes can be made even sharper with arrays of nanoscale apertures that compensate for optical aberrations more effectively than previous techniques.

The arrays allow for more accurate tracking of individual molecules in 3D, according to researchers at Stanford University.

Tracking how molecules move, form shapes and interact within the body's cells and neurons offers a powerful new view of key biological processes such as signaling, cell division and neuron communication, all of which impact people's health and susceptibility to disease.

The fraction of laser light transmitted through an array of subwavelength apertures is split into many beams, generating a starburst of fluorescence in a water droplet containing fluorescent dyes. The nanohole arrays at the side not covered by the droplet appear as small diffraction gratings. Courtesy of Optica.

With superresolution techniques, the location of a single molecule can typically be determined to within 10 nm. But even at such high resolutions, imperfections in the optical system can introduce aberrations that can significantly skew measurements, particularly in 3D. The resulting errors could mean the difference between interpreting two molecules as interacting or simply being close to each other.

While many researchers use fluorescent beads to calibrate 3D microscopes, doctoral candidate Alex von Diezmann took a different approach.

He created a 3D calibration standard out of a metal film with an array of <200-nm apertures regularly spaced 2.5 μm apart. Once the holes were filled with fluorescent dyes, the array could be used to calibrate for optical errors across the microscope's entire field of view, not just at a few select spots, as is possible using fluorescent beads. Using this technique, the researchers were able to correct aberrations of 50 to 100 nm to 25 nm.

"Prior to this, people had not explicitly worried about these aberrations," von Diezmann said. "The fact that we demonstrated the presence of field-dependent aberrations, and showed that they could degrade images, is an important part of this work."

The researchers studied the new calibration technique with double-helix and astigmatic point spread functions, two types of optical modification typically used to extract z-axis location. Although both point spread functions showed z-axis-related inaccuracies that created about a 20 percent error in the 3D measurements, the researchers corrected these aberrations using the 3D nanohole array.

The researchers are now applying the 3D calibration technique to all their single-molecule tracking and superresolution microscopy studies. For example, von Diezmann is using it to study protein localization in bacteria that measure only 2 μm in length. With the 3D calibration technique, he can accurately measure and track key signaling proteins in nanodomains 150- to 200-nm in size.

The work was led by Nobel laureate W. E. Moerner, who shared the 2014 Nobel Prize for Chemistry with Eric Betzig and Stefan W. Hell for pioneering work on superresolution microscopy.

"With the advent of superresolution imaging, we improved the resolution by a factor of five to 10 beyond the diffraction limit — from 200 nm down to 40 or even 10 nm," Moerner said. "This new world of greatly increased resolution brings a big transformation in how the optical system works."

The research was published in Optica (doi: 10.1364/optica.2.000985).