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LIONESS imaging reveals details of brain's complexity

25 Jun 2024

ISTA nanoscopy protocol includes modified STED super-resolution technique.

A project at the Institute of Science and Technology Austria (ISTA) has developed a novel imaging protocol to observe the brain's structure and dynamics.

Published in Nature Methods, the findings open up avenues for studying the functional nano-architecture of living brain tissue, according to the researchers.

"The human brain is the most sophisticated computational device with its network of around 86 billion neurons," commented ISTA.

"Understanding such complexity is a difficult task, and making progress requires technologies to unravel the tiny, complex interactions taking place in the brain at microscopic scales. Imaging is therefore an enabling tool in neuroscience."

The imaging and virtual reconstruction technology developed in Johann Danzl's High-Resolution Optical Imaging For Biology group at ISTA has been christened LIONESS, for Live Information Optimized Nanoscopy Enabling Saturated Segmentation.

LIONESS is a pipeline to image, reconstruct, and analyze live brain tissue with "a comprehensiveness and spatial resolution that was previously not achievable," said the project.

At present, 3D reconstruction of living brain tissue at the synapse level is hindered by insufficient resolution, inadequate signal to noise ratio (SNR), and prohibitive light burden in optical imaging. LIONESS has been designed to leverage optical modifications to super-resolution microscopy and combine them with deep-learning-based analysis, as a route past these obstacles.

"With LIONESS, for the first time, it is possible to get comprehensive, dense reconstruction of living brain tissue," said ISTA's Philipp Velicky.

"By imaging the tissue multiple times, LIONESS allows us to observe and measure the dynamic cellular biology in the brain take its course. The output is a reconstructed image of the cellular arrangements in three dimensions, with time making up the fourth dimension, as the sample can be imaged over minutes, hours, or days."

Fast and mild imaging conditions

The super-resolution microscopy element of LIONESS is based on the Nobel prize-winning STED (Stimulated Emission Depletion) technique, in which a second ring-shaped laser pulse is added to conventional fluorescence microscopy excitation, narrowing down the emitted signal from fluorophores to an area smaller than could be isolated otherwise.

But even STED has difficulty imaging entire volumes of brain tissue with resolution that matches the brain's complex 3D architecture, according to ISTA, because increasing resolution also entails a higher load of imaging light on the sample, which may damage or fry the living tissue.

"LIONESS has been developed for 'fast and mild' imaging conditions, thus keeping the sample alive," said ISTA. "The technique does so while providing isotropic super-resolution data, meaning that it is equally good in all three spatial dimensions, allowing visualization of the tissue's cellular components in 3D nanoscale resolved detail."

In trials, low-exposure/low-SNR and high-exposure/high-SNR 3D super-resolved image data were used to train deep neural image analysis networks, before LIONESS was then applied to living tissue from mouse hippocampi, a region densely packed with thin neurites, and human cerebral organoids.

Results showed that LIONESS "has the capacity to repeatedly retrieve both activity and dynamic structural information directly in the living state, with the potential to follow structural plasticity and determine structure–function relationships," commented the project in its paper.

"LIONESS opens up the decoding of complex, dynamic tissue architecture in living mammalian brain and other organs, and may ultimately challenge the way we think about the extent and significance of plasticity in the central nervous system."

Berkeley Nucleonics CorporationABTechOptikos Corporation CeNing Optics Co LtdSPECTROGON ABECOPTIKTRIOPTICS GmbH
© 2024 SPIE Europe
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