Adaptive Movement Compensation for In Vivo Imaging of Fast Cellular Dynamics within a Moving Tissue

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca2+ imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements

Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Understanding and treatment of spinal cord pathology is limited in part by a lack of longitudinal in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. Repeated imaging was routinely achieved for more than five weeks post-operatively with up to ten separate imaging sessions. We observed neither motor function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics following a laser-induced spinal cord lesion and observed massive microglia infiltration within one day along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions