Micro-two-photon Microscopy And Free-motion Mouse Neuroimaging

An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing in freely-behaving animals at the subcellular, cellular, circuit,and higher levels. In conjunction with fluorescent indicators, light microscopy has become a fundamental research tool in this quest, because it allows for direct visualization of neuronal activity at spatial and temporal scales covering many orders of magnitude. Single synapses are the elementary units of information transfer,processing, and storage, essential for the understanding of brain function and disease.The post-synaptic dendritic spines are sub-micron structures, buried deep within the brain,and operating at the millisecond scale. Owing to its intrinsic capacities for optical sectioning and deep tissue penetration, multi-photon microscopy has been the technique of choice for in vivo noninvasive optical brain imaging over the last two decades. Using benchtop two-photon microscopes (TPMs), morphological changes in spines, which are the neuronal basis of learning and memory, have been observed in vivo.Intricate spine activities in awake head-fixed mice are better resolved with state-of-the-art benchtop TPMs equipped with fast image acquisition (>15 Hz) and high excitation and photodetection efficiency. However, head-fixed animals are under physical constraint and emotional stress, and there is no a priori evidence demonstrating the equivalence of neuronal processes responding to virtual reality and to free exploration in real experimental environments. More importantly, many social behaviors, such as care-giving, mating, and fighting, are incompatible with the head-fixed paradigm[1],posing a fundamental limit to the understanding of brain activities in conditions close to the physiological environment.To meet these challenges, an attractive solution is to develop miniaturized microscopy to visualize the structural and functional dynamics in freely-moving and behaving animals over prolonged periods of time. Denk and colleagues built the first prototypical miniature TPM (mTPM) based on fiber-tip scanning in 2001, and this was followed by a decade of continuous effort by many other groups taking different approaches. However, we have been unable to find evidence of use of these mTPMs for actual follow-up biological applications, mainly due to two major limitations. First,none can image the most popular fluorescent probes like GCaMPs due to the lack of proper optic fibers to efficiently deliver 920-nm femtosecond laser pulses to the specimen without distortion. Second, mTPMs often underperform their theoretical resolution in in vivo experiments, presumably due to low sampling rates, aberrations of miniature graded index (GRIN) lenses, imperfection of miniature optics, and motion artifacts. At present, activity from neuronal structures smaller than dendrites cannot be robustly resolved by mTPMs despite a theoretical lateral resolution of ?1?m.Here we report the design, testing, and application of fast, high-resolution,miniaturized two-photon microscope (FHIRM-TPM) that resolves single-spine activity in freely-behaving animals. Weighing 2.15 g, the FHIRM-TPM is capable of imaging at high spatiotemporal resolution (0.64 ?m laterally and 3.35 ?m axially, 40 Hz at 256 x 256 pixels, FOV of 130 x 130 ?m2), and its micro-electromechanical systems (MEMS) scanner further confers fast free-line scanning and random-access imaging. By using a custom-designed hollow-core photonic crystal fiber (HC-PCF) to deliver 920-nm femtosecond laser pulses (named HC-920) with negligible nonlinear pulse-broadening, our FHIRM-TPM is able to image commonly used biosensors with a performance comparable to a benchtop TPM. In behavioral paradigms involving irregular, vigorous body and head movements (e.g., tail suspension, step-down from a stage, and social interaction), it enables robust and prolonged recording of activity from the somata, dendrites, and spines of cortical neurons labeled with GCaMP6f.Taken together, the FHIRM-TPM represents the next generation of miniature microscopy that fulfills the main promise of miniature microscopy for high-resolution brain imaging in freely-moving and behaving animals.