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Getting a bigger picture with superresolution microscopy

Chip-based illumination enables simple microscopy set-up and wide field of view

by Celia Henry Arnaud
April 27, 2017 | APPEARED IN VOLUME 95, ISSUE 18

Credit: Nat. Photonics
A direct STORM image acquired with chip-based illumination shows the interaction between actin (magenta) and the membrane (green) in liver sinusoidal endothelial cells. The inset shows the presence of actin between neighboring fenestrations.

Superresolution microscopy techniques allow researchers to observe objects tens of nanometers in size on or inside cells. But the methods can only keep an eye on patches 100 μm or less on a side at a time, making it difficult to image multiple cells simultaneously.

Now, by coupling a photonic chip with a standard optical microscope, researchers have achieved superresolution fluorescence microscopy with a simpler set-up and a wider field of view than conventional methods (Nat. Photonics 2017, DOI: 10.1038/nphoton.2017.55).

“You just need a basic microscope,” says Balpreet S. Ahluwalia of the Arctic University of Norway. “Our photonic chip technology can be retrofitted with any standard microscope to convert it into an optical nanoscope.” The chips allow Ahluwalia, Mark Schüttpelz of Bielefeld University, and coworkers to separate the illumination and detection pathways so the two don’t interfere with each other and enable the use of lenses with wider fields of view.

The waveguide chips direct laser light to the sample. Because the waveguides are made of high-refractive-index materials, the chips generate an intense evanescent field strong enough for the superresolution microscopy method called direct STORM (dSTORM).

The researchers integrated the chip with a standard optical microscope equipped with either a 20X or a 60X objective lens. With the 60X objective, the spatial resolution was better than 50 nm. With the 20X objective, the spatial resolution was only 138 nm, but the field of view was an extraordinarily large 0.5 mm x 0.5 mm. They used the system to image structures in liver cells.

So far, the chips are limited to total internal reflectance fluorescence, or TIRF, excitation. “This limits the use to imaging structures up to 150–200 nm away from the waveguide surface,” Ahluwalia says. But that’s also a benefit, he says, because TIRF illumination allows researchers to look at thin slices with very little background.

Suliana Manley, a superresolution imaging expert at the Swiss Federal Institute of Technology, Lausanne, says the work “represents a significant advance in making TIRF microscopy more accessible, since it doesn’t require an expensive TIRF objective.” For superresolution imaging, membrane biologists are most likely to benefit from the technology, she says.

“Anyone interested in large-field-of-view imaging will benefit from this,” Ahluwalia says. “Using our photonic chip, we can generate with a slight reduction in spatial resolution a super-resolved image over approximately 100x larger area than what can be achieved today with commercial dSTORM microscopy.”



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