Zhang, Y.; Bai, L.; Wang, X.; Zhao, Y.; Zhang, T.; Ye, L.; Du, X.; Zhang, Z.; Du, J.; Wang, K.

Neurons are best studied in their native working states in which their functional and morphological dynamics support animals\' natural behaviors. Super-resolution microscopy can potentially reveal these dynamics in much higher details but has been challenging in behaving animals due to severe motion artifacts. Here, we report multiplexed line-scanning structured illumination microscopy, which could tolerate motions of up to 50 um/s while achieving 150 and 100 nm lateral resolutions in its linear and nonlinear forms respectively over large fields-of-view. We continuously imaged the dynamics of spinules in dendritic spines and axonal boutons volumetrically over thousands of frames and tens of minutes in mouse brains during sleep-wake cycles. Super-resolution imaging of axonal boutons revealed their prevalent spinule dynamics on a scale of seconds. Simultaneous two-color imaging further enabled analyses of the correlation between the spatial distributions of diverse PSD-95 clusters and the structural dynamics of dendrites in the brains of awake mice.