Structured illumination assists imaging of mouse brain

05 Apr 2022

SIMscope3D device reaches deeper to view cells and circuits.

Miniature microscopes able to be mounted on living animals are a major topic of research, thanks to their potential for imaging neural activity in vivo.

Current examples include the MINI2P probe, specifically designed to bring the advantages of 2-photon imaging and its ability to eliminate out-of-focus light to the imaging of mouse brains, by miniaturizing the necessary optics architecture.

A project at the University of Colorado Boulder has now developed a miniature fluorescence microscope intended for the imaging of brain structures which employs structured illumination as a means to enhance performance, said to be the first of its kind.

Described in Biomedical Optics Express, the team's SIMscope3D device is designed to allow deeper imaging than miniature widefield microscopes can achieve, to reveal details of how brain cells and circuits operate.

In particular, the Boulder study was intended to tackle challenges associated with removal of scattered light that still exist with modified 3D miniscopes in these applications, particularly when it comes to identifying structural features.

"Developing new treatments for neurological disorders requires understanding the brain at the cellular and circuit-level," said Emily Gibson from the University of Colorado Anschutz Medical Campus. "New optical imaging tools, particularly those that can image deep into brain tissue like the microscope our team developed, are important for achieving this goal."

Using some form of structured light, such as a grid pattern of illumination, in conjunction with image reconstruction techniques can offer a route to optical sectioning, although the optical components needed can be unsuitable for the small lightweight platforms envisaged in live animal studies.

The Boulder solution involved using a coherent fiber bundle to deliver the spatially patterned light to the miniature microscope objective, along with a tunable electrowetting lens that allows 3D visualization of brain structures by changing the microscope’s focal depth without requiring any moving parts.

Understanding multiple sclerosis

The researchers also integrated a CMOS camera directly into the microscope, enabling imaging with high lateral resolution while avoiding artifacts that might be induced if the images traveled through the fiber bundle. Using an LED light source, the new microscope can produce sharp contrast even when imaging deeply into highly scattering tissue, according to the team.

"The components chosen are aimed towards providing high contrast and resolution, which makes this miniature microscope different from others that have been designed for imaging neural activity from cells where higher contrast and resolution is not required," noted the team in its paper.

In trials, the system imaged oligodendrocytes and microglia labeled with a fluorescent protein in mice that were awake but placed in a device that kept their head stationary, achieving axial resolution of 18 microns. Damage to oligodendrocytes is an important aspect of multiple sclerosis, and improved imaging of such cells could be important in treatment of the disease.

"We used our miniature microscope to record a time series of glial cell dynamics in awake mice at depths up to 120 microns in the brain," said Boulder's Omkar Supekar. "Scientists don’t fully understand exactly how these cells work or their repair processes. Our microscope opens the possibility of long-term studies examining how these cells migrate and are repaired."