Misc

The flow of cerebrospinal fluid is considered to be a critical factor in the clearance of wastes in the brain. The dynamics of fluid has been investigated by several experimental methods such as fluorescence microscopy and magnetic resonance imaging. However, because probes used for these imagings are much larger compared to water itself, the dynamics of the fluid have been poorly understood. Here, we applied a multimodal multiphoton imaging system to the living brain tissue. Combining stimulated Raman scattering and two-photon fluorescent imaging, the system enables us to visualize spatiotemporal dynamics of deuterated water and fluorescent dyes simultaneously at a cellular level. We demonstrate that deuterated water diffuses faster than fluorescent dyes in the brain tissue. Detailed analysis reveals deuterated water rapidly exchanges inside and outside of cells, whereas fluorescent dyes only diffuse through extracellular spaces. Furthermore, we find that the dynamics of deuterated water is robust to changes under physiological and pathophysiological conditions; there is little change in the spatiotemporal dynamics of deuterated water during development and ischemia whereas fluorescent dyes are severely affected. Thus, our new approach reveals unique properties of the dynamics of the fluid in the living brain tissue.