High resolution functional imaging of neuronal activity by wave front shaping of ultra fast pulses – WaveFrontImag

Voltage and calcium imaging can revolutionise our understanding of how neuronal cells and circuits process and store information in the living brain. To explore the mechanisms underlying synaptic transmission, membrane potential and calcium transients must be measured from neuronal dendrites and spines where synaptic contacts are formed. Today voltage and calcium imaging are efficiently implemented using either wide-field or local two-photon scanning illumination. The first strategy, combined with camera detection, permits multisite measurements but is limited in spatial resolution and penetration depth. The second strategy allows in depth imaging with diffraction limited excitation volumes but is limited in temporal resolution for multisite recording. Here we propose to develop a new method combining the advantages of the two existing strategies. Precise sculpting of the excitation shape and intensity will be achieved by wave front shaping of ultra-fast laser pulses. Precisely, we will use the technique of generalised phase contrast combined with temporal focusing to illuminate specific cellular or subcellular regions in combination with a novel sCMOS camera permitting high frame rate acquisition. In contrast to 1-photon wide-field illumination, axially resolved patterned excitation will minimise signal degradation arising from nonspecific fluorescence scattered from nearby labelled structures. In contrast to 2-photon scanning approaches, the use of parallel excitation will permit improving the temporal resolution. Finally, to illuminate with high light intensity the dimmest regions without saturating the brightest ones, we will use illumination patterns with intensity gradients thus achieving uniform resting fluorescence and optimal signal to noise ratio. We will validate this new approach on proof of principle experiments using well established electrophysiology protocols in neurons from brain slices individually loaded with calcium indicators and voltage sensitive dyes. In the last part of the project we will use this new approach to investigate membrane potential and calcium signals in cerebellar Purkinje neurons focussing on signals in dendrites and spines. In particular, we will focus on the recording of fast calcium currents mediated by dendritic voltage-gated Ca2+ channels using a recently developed method based on ultra-fast calcium imaging with low-affinity Ca2+ indicators. These measurements will shed new light on the role of dendritic voltage-gated calcium channels in synaptic signalling in the cerebellum. The commercial exploitation of the technology developed in this project will be done through existing industrial collaborations. This project will be partly supported by the LabEx “Ion Channels Science and Therapeutics” and by the National Infrastructure France Bio-Imaging.