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Mirror improves STED resolution

22 Jun 2016

Interference of local electromagnetic field delivers six-fold improvement in axial resolution with no increase in laser power.

An inter-continental team of researchers has found a relatively simple way to improve the resolution of stimulated emission depletion (STED) microscopy – one of the super-resolution techniques that won a Nobel prize for its inventors in 2014.

The scientists, based at Georgia Tech in the US, Australia’s Centre of Excellence for Nanoscale Biophotonics (CNBP) in Sydney, and Peking University in China, replaced the traditional microscope slide with a small, carefully aligned mirror.

Called mirror-enhanced axial-narrowing super-resolution (MEANS) microscopy, their technique exploits optical interference to improve STED axial resolution by a factor of six, and lateral resolution by a factor of two.

Discussing the details in the Nature-published journal Light: Science & Applications, the team writes that the improved resolution required no additional complexity in the optical system (they used a commercial STED microscope from Leica), and allowed them to visualize elements of a virus that would not be possible with standard STED.

Sydney professor Jin Dayong, from the CNBP’s Advanced Cytometry Labs, said: “This simple technology is allowing us to see the details of cells that have never been seen before.

“A single cell is about 10 micrometers; inside that is a nuclear core about 5 micrometers, and inside that are tiny holes, called the 'nuclear pore complex,' that as a gate regulates the messenger bio-molecules, but measure between one-fiftieth and one-twentieth of a micrometer. With this super-resolution microscopy we are able to see the details of those tiny holes.”

Adjustable thickness
In their journal article Jin and colleagues detail the specific nature of the mirror that is needed for the MEANS technique. They report:

“The mirror should be a first-surface mirror, with a protective SiO2 coating, and an adjustable thickness so that the constructive interference with a high-numerical aperture objective can occur within the specimen.” The collaboration tested thicknesses of 50, 100, 150 and 200  nm.

Thanks to the silica layer, cells can grow normally on the mirror’s surface, they explain, adding that a coverslip can be applied to seal the specimen. “We have a custom-made mirror holder that is the same size as a microscope slide so that the mirror-backed specimen can be placed easily on any commercial confocal microscope.”

The improved axial sectioning and super-resolution capability means that, for the very first time, optical microscopy revealed the inner ring structure of a nuclear pore complex, as well as the tubular structure of a single, rod-shaped virus particle, claims the team.

Power problem
One of the general criticisms of STED microscopy is the high photon flux needed to generate the super-resolution images, which can be damaging to biological samples. Jin and colleagues point out that with MEANS, no additional laser power is required to improve either the lateral or axial resolution.

They achieved a best resolution of 19 nm using the 592 nm wavelength depletion laser provided with the Leica microscope, delivering 60 mW to the samples.

“To the best of our knowledge, this resolution is among the best recorded for STED super-resolution in biological specimens,” they claim in their paper. “The resolution improvement is largely because two-fold resolution enhancement can be obtained without increasing the depletion laser power.”

They add that the MEANS approach is compatible with a variety of other confocal imaging techniques, including two-photon, spinning disk and laser scanning microscopy.

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