Light sheet fluorescence microscopy breakthrough
Image: Federico Gasparoli and Kishan Dholakia with the newly developed three-photon light-sheet fluorescence microscope.
Claiming a world first, Scotland-based researchers have developed three-photon absorption to increase the imaging depth of light-sheet fluorescence microscopy.
By combining this absorption process with light sheet microscopy, Professor Kishan Dholakia from the University of St. Andrews, and colleagues, clearly imaged a cultured cellular spheroid, some 450 microns in diameter.
“This demonstration is very important as it addresses an unmet need of better imaging at depth, which could help scientists gain better data about biological processes,” says Dholakia. “This approach could be especially useful for neuroscience and developmental biology studies and might find application in imaging multiple samples in an automated way for drug discovery.”
“We expect three-photon light-sheet fluorescence microscopy to make a large impact on imaging the brain in rodents such as mice and rats, where it could be used to capture very wide-field images with subcellular resolution,” adds Dholakia's colleague, Adrià Escobet-Montalbán.
Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low-photodamage technique for fast volumetric imaging of biological samples.
However, as Dholakia explain in Optics Letters, he and colleagues wanted to extend this imaging modality to the three-photon regime, enhancing its penetration depth.
To compare three-photon and two-photon light-sheet fluorescence microscopy, Dholakia and colleagues used a standard optical setup with a 100 nm femtosecond pulsed laser, as traditionally used for two-photon excitation.
They then imaged spheroids of human embryonic kidney cells using two-photon and three-photon excitation.
3D rendering of a human embryonic kidney.
Results revealed that near the spheroid’s surface, both imaging modalities performed similarly.
However, at the far side of the spheroid, the image quality for the three-photon light-sheet fluorescence microscopy preserved image contrast while the quality of the two-photon image declined considerably.
To further improve the depth imaging and field of view, the researchers experimented with changing the light intensity profile of the laser from that of a traditional solid Gaussian laser beam to a Bessel beam.
“Bessel beams can be used in two-photon light-sheet mode but may yield potential artifacts due to their concentric rings,” explains Federico Gasparoli, also from the St Andrew's team.
"However, for the first time, we show numerically and experimentally that these problems are suppressed in three-photon light-sheet fluorescence microscopy and that the beam goes even deeper, making this approach very attractive,” he adds.
To increase imaging depth even more, the researchers now plan to use laser systems at longer wavelengths, specifically designed for three-photon absorption.
In parallel, the researchers are developing ways to detect the light emitted from fluorescent labels deep inside samples.
Research is published in Optics Letters.