image: This is the Schematic diagram and the performance of skull optical clearing window (SOCW) technique. (a) Four steps: immobilization, skull optical clearing, cortical imaging, and recovery. (b) A custom-built head immobilization device consisting of a skull-holder and a custom-built plate is used to reduce motion artifact during imaging. (c) Anatomical structure of mouse skull. (d) Schematic of the SOCW. A layer of plastic wrap is placed over the cleared skull to separate the water immersion objective from the optical clearing agent (OCA). (e) Representative fluorescence images of the dendrites through the skull, before and after skull optical clearing. We found that the image quality was considerably improved and that it was sufficient for imaging the dendritic spines through the cleared skull. (f) The imaging depth through the SOCW. Orthogonal (x-z) projections of dendrites, before and after skull optical clearing, demonstrating that the depth enhances obviously after clearing (the imaging parameter and data processing were the same). (g-h) The repeatability of SOCW technique. Repeated imaging of the dendrites (g) and spines (h) of Thy1-YFP neurons obtained over 1-d interval. Scale bar, 10 μm. view more
Credit: Skull optical clearing window for in vivo imaging of the mouse cortex at synaptic resolution. Yan-Jie Zhao, Ting-Ting Yu, Chao Zhang, Zhao Li, Qing-Ming Luo, Tong-Hui Xu & Dan Zhu. <i>Light: Science & Applications</i> volume 7, page 17153 (2018) doi:10.1038/lsa.2017.153
A non-invasive approach for creating an optical window in the skull to enable the brains of living mice to be imaged has been demonstrated. Prof. Dan Zhu and coworkers from Huazhong University of Science and Technology, China, tested the use of optical clearing agents (OCAs) that they applied to the bare skulls (hair and skin removed) of living mice. After treatment with OCAs, the skull becomes transparent within minutes, and thus forming a visible window of the cortex. Combined with two-photon microscopy, this technique allows imaging of the fine structures of neurons, glia and the microvasculature in the mouse brain. Given its easy handling, safety, repeatability and excellent performance, this method has promise in neuroscience research. The research has been published in journal Light: Science and Applications on February 23rd, an open access journal from Nature Publishing Group.
Chronic observation and manipulation of cells in the cortex is cranial to studies of brain structure and function. However, the strong scattering caused by the skull over the cortex limits the penetration depth of light in tissues, and thus hinders the observation of fluorescently labeled neuronal structures and microvasculature. To overcome this obstacle, various cranial window methods were developed, including the open-skull glass window, the thinned-skull cranial window, and their variants. But these methods present limitations. The tissue optical clearing technique can reduce the scattering of tissues, which has become an important tool for the applications of optical imaging in biomedical research. However, the current optical clearing method is widely used in the ex vivo studies of various tissues and organs, and these are few studies on that how to make the living tissues transparent.
Prof. Dan Zhu firstly proposed the study of in vivo optical clearing technique. In the early stage, she was focused on the research of different types of skin tissue. Prof. Tonghui Xu, who is Prof. Dan Zhu's colleague, has been engaged in the research of cortical neuroimaging in mice, and for in vivo cortical imaging the turbid skull becomes a great bottleneck. After communicating with Prof. Tonghui Xu, Prof. Dan Zhu realized the great need of neuroscientists and began to engage in the research on optical clearing of skull tissue. After six years of hard work, they developed an effective, safe and switchable skull optical clearing window. Through this window, the image contrast and imaging depth are significantly improved, and it can realized the imaging of the cortical structures at synaptic resolution. This technique holds great promise for the studies of brain structure and function in physiological or disease states.
This study was supported by the National Natural Science Foundation of China (Grants Nos. 91232710, 31571002), the Science Fund for Creative Research Groups (Grant No. 61721092), and the Director Fund of WNLO. Prof. Dan Zhu and Tonghui Xu are the corresponding authors, Prof. Qingming Luo participated in the guidance. Yanjie Zhao (the student of Prof. Dan Zhu) is the first author, Dr. Tingting Yu, Chao Zhang and Zhao Li are the co-authors.
The paper link: http://www.nature.com/articles/lsa2017153
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