Accurate classification of major brain cell types using in vivo imaging and neural network processing.

Comprehensive analysis of tissue cell type composition using microscopic techniques has primarily been confined to ex vivo approaches. Here, we introduce NuCLear (Nucleus-instructed tissue composition using deep learning), an approach combining in vivo two-photon imaging of histone 2B-eGFP-labeled cell nuclei with subsequent deep learning-based identification of cell types from structural features of the respective cell nuclei. Using NuCLear, we were able to classify almost all cells per imaging volume in the secondary motor cortex of the mouse brain (0.25 mm3 containing approximately 25,000 cells) and to identify their position in 3D space in a noninvasive manner using only a single label throughout multiple imaging sessions. Twelve weeks after baseline, cell numbers did not change yet astrocytic nuclei significantly decreased in size. NuCLear opens a window to study changes in relative density and location of different cell types in the brains of individual mice over extended time periods, enabling comprehensive studies of changes in cell type composition in physiological and pathophysiological conditions.