Fast High-resolution Miniature Two-photon Microscopy for Brain Imaging in Freely-behaving Mice at the Single-spine Level

Recent developments in miniaturized microscopes have furthered the quest to visualize brain activities and structural dynamics in animals engaged in self-determined behaviors. However, it remains an unmet challenge to resolve activity at single dendritic spines, the elemental units underlying neuronal computation, in freely-behaving animals. Here, we report the design, testing, and application of a fast, high-resolution, miniaturized two-photon microscope (FIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a new type of hollow-core photonic crystal fiber to deliver 920-nm femtosecond laser pulses, the FIRM-TPM is capable of imaging commonly used biosensors at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels). Its micro-electromechanical systems scanner also enables random-access capability and free-line scanning at up to 10,000 Hz. It compares favorably with benchtop two-photon microscopy and miniature wide-field fluorescence microscopy in the structural and functional imaging of Thy1-GFP- or GCaMP6f-labeled neurons. Further, we demonstrate its unique application and robustness with hour-long recording of neuronal activities down to the level of spines in mice experiencing vigorous body and head movements or engaging in social interaction. Thus, our new generation miniature microscope provides neuroscientists the long-sought tool-of-choice for imaging the brain at the synaptic level in freely-behaving animals.