Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

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Change history

Corrected online 20 June 2017

In the version of this article initially published online, the affiliation for Aimin Wang was incorrectly listed as Department of Neurobiology, Institute of Basic Medical Sciences, Beijing, China. The correct affiliation is State Key Laboratory of Advanced Optical Communication System and Networks, School of Electronics Engineering and Computer Science, Peking University, Beijing, China. The error has been corrected in the print, PDF and HTML versions of this article.