Visualizing astrocytes in the deep mouse brain in vivo

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3-photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength-dependent 3-photon action cross section (ησ ₃ ) measurement of SR101. 3-photon fluorescence imaging of the SR101-labeled vasculature enabled an imaging depth of 1340-μm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal-to-background ratio limit encountered in 2-photon fluorescence imaging. Visualization of astrocytes 910 μm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2-photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101-labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.