HKUST images mouse cortex at 'unprecedented depth'

22 Jun 2022

Combination of three-photon microscopy and adaptive optics reveals details of the brain.

The latest advance in mouse brain imaging comes from Hong Kong University of Science and Technology (HKUST), combining three-photon and adaptive optics techniques.

HKUST has demonstrated in vivo imaging of fine neuronal structures in mouse cortex through the intact skull, at an "unprecedented depth" of 750 microns, according to the project. This should allow non-invasive high-resolution microscopy of the cortex and facilitate study of the living brain.

"Over recent decades, great effort has been focused on developing novel techniques for in vivo imaging of the intact brain," commented the project team. "However, none of the prevalent technologies, including ultrasound imaging, positron emission tomography and MRI, provides sufficient spatial resolution to visualize biological structures at the subcellular level."

The HKUST approach combines three-photon microscopy (3PM) with two forms of adaptive optics (AO), demonstrating fast measurements and the correction of both low-order and high-order aberrations in tissue at depth.

Both technologies are known to be potentially significant in imaging neurons and biomolecules deeper within in vivo small animal brains. Recent breakthroughs have included a platform developed at Cornell Neurotech, using 3PM to trigger fluorophores deeper within the tissues of adult zebra fish than two-photon methods achieve.

In 2021 a project at Germany's EMBL lab developed a platform combining 3PM and AO to image deep cortical spines and sub-cortical dendrites in a mouse brain, describing it as "the first time these techniques have been combined."

According to HKUST, its new AO-3PM platform makes use of two AO techniques: direct focus sensing with phase-sensitive detection, and conjugate adaptive optics (CAO) with remote focusing.

"The guide star signal is coded and then decoded in the aberration measurement, to achieve AO correction of aberrations," commented the project. "These enable the accurate measurement of the aberrant electric-field point-spread function of a laser in tissue and the fast correction of the aberration over a large imaging volume in the brain."

Widen the understanding of the living brain

In trials, the conjugate adaptive optics configuration with remote focusing enabled in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 microns below the pia matter, the membrane surrounding the brain, enabling near-non-invasive high-resolution microscopy of the cortex.

Taking advantage of the tight focus provided by AO, the team went on to demonstrate the capability of its AO-3PM approach to guide precise laser microsurgery, and investigate post-operative microglial dynamics in the cortex through the intact skull.

"Functional calcium imaging with high sensitivity and high-precision laser-mediated microsurgery through the intact skull were also demonstrated," noted the project in its Nature Biotechnology paper. "Moreover, we achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 millimeters below the pia within the brain."

HKUST believes the results point to a new tool for experimental biology, demonstrating that AO-3PM technology holds great potential to advance imaging techniques and facilitate study of living brain.

"It is truly remarkable what this state-of-the-art AO-3PM system can achieve," said HKUST's Nancy Ip. "The high performance and unparalleled accuracy of this advanced deep-brain imaging technology will substantially widen our understanding of living brain with optimal physiological representation."