Researchers from Tsinghua University have developed a way to image multiple areas of the brain at the same time, both on the brain’s surface and deep inside the brain. Since brain processes often span multiple regions of the brain, researchers say the new approach could lead to a more complete understanding of how the brain works in health and disease. To allow simultaneous imaging of multiple areas and depths in the brain, the researchers implanted glass windows in the heads of living mice. They accomplished this by removing part of the skull and implanting two separate windows into the brain. The first window was a glass plug 2 mm wide and 0.9 mm tall. The second window was a microprism in the shape of a 1-mm cube. Although the researchers could use either window to image the brain at different depths, they found that the glass plug provided a more detailed view of the structures near horizontal surfaces, and the microprism showed the more longitudinal structures of deeper brain tissues with greater clarity. Using these windows into the mouse brain, the researchers were able to take detailed, simultaneous images of cell structures up to 1 mm deep and with a 1-mm-wide field of view. The researchers used real-time uItralarge-scale high-resolution macroscopy (RUSH), which is an imaging technique that combines a large field of view and high resolution, to acquire images through the windows. RUSH provided the researchers with a 1-cm field of view, video-rate acquisition, and 800-nm resolution. To test their dual-window approach, the Tsinghua researchers imaged microglia cells (a type of immune cell found in the brain and central nervous system) in the superficial cortex and in the hippocampus of living mice. Images showed the cellular structures of both regions in detail, even though the cells in the hippocampus were about 0.9 mm deeper than those in the superficial cortex. The implanted microprism provided a longitudinal view of the cortex from a depth of 0 to 1 mm. The field of view for both images was at least 1 mm across. While wide-field microscopes are frequently used for brain imaging, they typically can image only to a depth of 200 to 300 µm. The researchers used their custom-built cranial windows to overcome this limitation and allow simultaneous imaging of deep structures and those near the surface. The researchers said that the key advantage of their method is the ability to simultaneously observe the near-whole superficial cortex while observing the deep brain tissue horizontally or longitudinally, with a brain-wide field of view, video-rate acquisition, and cellular resolution. Implanting the glass cranial windows is low-cost compared to using a GRIN lens, and the dual-windows method is compatible with other wide-field microscopes. Many neurological diseases are associated with more than one region of the brain. For example, memory involves interaction between the cortex and the hippocampus. Spontaneous epileptiform activity can spread from the lesion site to the whole cortex and even to the hippocampus in seconds. “We think this method could promote research of the brain and diseases and bring great discoveries,” researcher Chaowei Zhuang said. “In future work, we will continue to develop new neural imaging techniques with higher throughput, higher signal-to-noise ratio, and lower cost. Secondly, [we will] explore new algorithms to understand the insight behind the observed cortex-wide neural dynamics. Finally, we will introduce the method to a wide range of biomedical applications, which require simultaneous observation of cellular dynamics at different depth, such as the study of epilepsy.” The research will be presented at the virtual OSA Imaging and Applied Optics Congress and Optical Sensors and Sensing Congress to be held July 19-23, 2021. The researchers will demonstrate the functional imaging results of neurons in mouse brains during the presentation.