Super-resolution imaging of fast morphological dynamics of neurons in behaving animals

Neurons are best studied in their native states in which their functional and morphological dynamics support animals’ natural behaviors. Super-resolution microscopy can potentially reveal these dynamics in higher details but has been challenging in behaving animals due to severe motion artifacts. Here we report multiplexed, line-scanning, structured illumination microscopy, which can tolerate motion of up to 50 μm s⁻¹ while achieving 150-nm and 100-nm lateral resolutions in its linear and nonlinear forms, respectively. We continuously imaged the dynamics of spinules in dendritic spines and axonal boutons volumetrically over thousands of frames and tens of minutes in head-fixed mouse brains during sleep–wake cycles. Super-resolution imaging of axonal boutons revealed spinule dynamics on a scale of seconds. Simultaneous two-color imaging further enabled analyses of the spatial distributions of diverse PSD-95 clusters and opened up possibilities to study their correlations with the structural dynamics of dendrites in the brains of head-fixed awake mice.