ADAPT: Adaptive and predictive arbitrary point scanning two photon tomography

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Multiphoton microscopy is today unrivalled among other optical approaches in intravital imaging, as it allows imaging of labelled structures and proteins while providing large optical penetration depth with sub-cellular axial resolution, keeping comparatively low photobleaching levels. These advantages, along with the possibility of exciting genetically encoded calcium and voltage indicators, have made multiphoton microscopy the method of choice for neuronal studies in rodents to decipher brain function and understand neuronal diseases. Optical sampling at high speeds is of paramount importance for accurately capturing neural activity, as the characteristic timescales for action potentials are of the order of a few milliseconds, while Ca transients correlate in the 100 ms range. Current imaging technologies do not sufficiently combine fast recording rates with volumetric sampling, which is equally important to fully understand the inherently three-dimensional neuronal circuitry. With this WEAVE project, we aim to advance and tailor the spectro-temporal laser imaging by diffractive excitation technique previously introduced by one partner to the challenges of neurophotonics by: i) acquiring volumetric imaging of layered neuronal structures (cortex, intestine) at the inherent timescales of their functional processes (20Hz volume rate); ii) developing a dedicated near infrared source to image and benefit from the matured GCaMP family of fluorescence proteins; iii) analyzing and optimizing signal yield and sample damage introducing rapid adaptive scanning and sampling protocols; iv) correcting wavefront distortions and accounting for specimen movements by employing nonlinear fiduciary markers embedded in tissue depth. To achieve these goals, the ADAPT research partners have joined in a synergetic WEAVE consortium with laser physicist and inventor of spectro-temporal Multiphoton imaging Sebastian Karpf (GER) to build a volumetric imaging system at 940 nm that is perfectly tuned to image GFP-like molecules at 20 Hz volumetric imaging; nanophotonics and nonlinear optics expert Luigi Bonacina (CH) to advance, assess, and translate the disruptive technology to adaptive and targeted sampling exploiting the active modulation of the source and bespoke solutions for three-dimensional scanning. Further, LB will set-up a dedicated SHG detection channel to collect information from a nonlinear guide star to counteract propagation-induced wavefront distortions and account for sample movements (heartbeat, breathing). Finally, neurophysiology expert Pieter Vanden Berghe (BE) will study together with LB the advantages brought forth by the high-speed, advanced scanning protocols to optimize and investigate signal yield and sample damage as compared to state-of-the-art femtosecond systems. PV will also apply the ADAPT technology to volumetric neuronal activity imaging using GCaMP to push the current boundaries of neuronal intravital imaging.