Researchers from the University of Colorado Boulder, the University of Colorado Anschutz Medical Campus, and Arizona State University have developed a head-mounted, lightweight, fluorescence microscope that provides full 3D imaging and enhanced contrast in scattering tissue through optical sectioning. By imaging deeper into the brain than any previous miniature widefield microscope, the researchers believe their device could help improve the ability to observe neural circuits and their function. The miniature microscope, called the SIMscope3D, is the first miniature microscope to use structured illumination to remove out-of-focus and scattered light, the researchers said. The SIMscope3D performs volumetric imaging by using an imaging fiber to deliver spatially patterned light to the miniature microscope objective. This process also removes out-of-focus light, through an optical sectioning process similar to that found in two-photon microscopy, but without the complex components or expensive laser. In tests, the miniature microscope obtained optical sectioning with an axial resolution of 18 µm. Structured illumination allows the device to image as deep as 260 µm. The SIMscope3D uses a digital micromirror device to create a structured illumination pattern that is conveyed to the imaging plane through a coherent fiber bundle. A CMOS camera with a 2.2-µm pixel size is integrated into the microscope, enabling high-lateral-resolution images while preventing the artifacts that might occur if the images were to travel through the fiber bundle. The microscope includes a compact, tunable electrowetting lens that allows 3D visualization of brain structures by changing the microscope’s focal depth, without requiring any moving parts. The electrowetting axial scanning element provides depth scanning of up to 550 µm into the sample. The SIMscope3D images fluorescence emitted from tissue or fluorescent tags after the sample is exposed to certain wavelengths. Using the microscope, the researchers imaged brain tissue of glial cells labeled with a fluorescent protein in mice that were awake, but placed in a device that immobilized their heads. “We used our miniature microscope to record a time series of glial cell dynamics in awake mice at depths up to 120 µm in the brain,” researcher Omkar Supekar said. “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.” In addition to demonstrating background-free 3D imaging in awake mice, the researchers demonstrated volumetric imaging at depths up to 260 µm. In previous head-mounted, widefield, fluorescence microscopes, light scattered by tissue has blocked the ability to image deep into the brain. Miniature two-photon microscopes can overcome this drawback by eliminating out-of-focus light in each focal plane. However, these microscopes typically require expensive pulsed lasers and complex mechanical scanning components. Using an LED light source, the SIMscope3D can produce sharp contrast even when imaging deeply into highly scattering tissue. The microscope costs less and can use higher frame rates than a two-photon miniature microscope. With these features, the SIMscope3D can support the investigation of dynamic neural structures and functions in behaving animals. Detailed time-lapse images of brain cells taken with the SIMscope3D could lead to new insights into neurological disorders, such as multiple sclerosis. “Developing new treatments for neurological disorders requires understanding the brain at the cellular and circuit-level,” professor Emily Gibson said. “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.” Researchers developed a head-mounted microscope that uses structured illumination to remove out-of-focus light with optical sectioning. This enables deep imaging while also enhancing image contrast in scattering tissue. Courtesy of Omkar D. Supekar/University of Colorado Boulder, and Emily Gibson/University of Colorado Anschutz Medical Campus. The researchers are currently working to improve the microscope’s acquisition speed and weight. With minor upgrades, the microscope will be able to image faster dynamics, such as neuronal electrical activity, while the subject animal performs different tasks. The researchers said that the microscope could be easily developed into a commercial system for use in neuroscience labs, since it does not require expensive components. “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,” Supekar said. The research was published in Biomedical Optics Express (www.doi.org/10.1364/BOE.449533).