Researchers have developed a miniature microscope designed for high-resolution 3D images inside the brains of living mice. By imaging deeper into the brain than before with miniature, wide-field microscopes, the new lightweight microscope could help scientists better understand how brain cells and circuits work.
“With further development, our microscope will be able to image neural activity over time while an animal is in a naturalistic environment or performing different tasks,” said lead author Omkar Supekar of the University of Colorado at Boulder. “We show that it can be used to study cells that play an important role in neurological disorders such as multiple sclerosis.”
In the magazine Optica Publishing Group Express Biomedical Optics, the researchers describe their new SIMscope3D, which images the fluorescence emitted by tissues or fluorescent labels after the sample is exposed to certain wavelengths of light. The new device is the first miniature microscope to use structured illumination to eliminate blurry and scattered light, enabling imaging as deep as 260 microns on fixed brain tissue with an LED light source.
“Developing new treatments for neurological disorders requires understanding the brain at the cellular and circuit level,” said Emily Gibson, research team leader, of the University of Colorado’s Anschutz Medical Campus. “New optical imaging tools, especially those that can image deep into brain tissue like the microscope developed by our team, are important in achieving this goal.”
Head-mounted microscopes are used to image the brains of small rodents through transparent windows implanted in their skulls. Researchers have already developed wide-field, head-mounted fluorescence microscopes, but tissue-scattered light prevents imaging deep into the brain. Miniature two-photon microscopes can overcome this drawback by eliminating out-of-focus light in each focal plane – a process known as optical sectioning – but generally require expensive pulsed lasers and complex mechanical scanning components.
To design the new microscope, Andrew Sias, Sean Hansen, Gabriel Martinez, and Emily Gibson of the Department of Bioengineering at the University of Colorado’s Anschutz Medical Campus; Douglas Shepherd of the Department of Physics at Arizona State University; Omkar Supekar and Juliet Gopinath of the Department of Electrical, Computer, and Power Engineering, and Victor Bright of the Department of Mechanical Engineering at the University of Colorado Boulder worked closely with neuroscientists Graham Peet, Diego Restrepo, and Ethan Hughes of the Department of Cellular Engineering and developmental. Biology and Xiaoyu Peng and Cristin Welle from the Department of Physiology and Biophysics at the University of Colorado’s Anschutz Medical Campus to optimize it for the study of the brain.
Volumetric imaging is performed by using an imaging fiber to deliver spatially patterned light to the miniature microscope objective. This process also removes out-of-focus light, allowing for an optical cut similar to that achieved with two-photon approaches, but without the complex components or expensive laser.
The microscope includes a compact, tunable electrowetting lens that allows 3D visualization of brain structures by changing the focal depth of the microscope without the need for moving parts. The researchers also integrated a CMOS camera directly into the microscope. This allows imaging with high lateral resolution while avoiding artifacts that could be induced if the images passed through the fiber bundle. Using an LED light source, the new microscope can produce sharp contrast even when imaging deep in highly scattering tissue.
Capture glial cells
The researchers demonstrated their new system by imaging oligodendrocytes and microglia labeled with a fluorescent protein in mice that were awake but placed in a device that held their heads still. In people with multiple sclerosis, the oligodendrocytes, which form an insulating layer around the axons, are destroyed. This slows down connections in the brain, leading to impaired vision, motor skills, and other problems.
“We used our miniature microscope to record a time series of glial cell dynamics in awake mice at depths of up to 120 microns in the brain,” Supekar said. “Scientists don’t understand exactly how these cells work or their repair processes. Our microscope opens up the possibility of long-term studies examining how these cells migrate and are repaired.”
The researchers are now working to improve the acquisition speed and weight of the microscope. With minor upgrades, the microscope will be able to image faster dynamics, such as neuronal electrical activity, while the mouse performs different tasks. The researchers say that because the microscope doesn’t require expensive components, it could be easily expanded into a commercial system for use in neuroscience labs.
Paper: OD Supekar, A. Sias, SR Hansen, G. Martinez, GC Peet, X. Peng, VM Bright, EG Hughes, D. Restrepo, DP Shepherd, CG Welle, JT Gopinath, EA Gibson, “Miniature Structured Illumination Microscope for In vivo 3D imaging of brain structures with optical sectioning,” Biomedical. Opt. Express, volume 13, number 4, p. 2530-2541 (2022). DOI: doi.org/10.1364/BOE.449533