COMBINING FAST VOLUMETRIC IMAGING AND TWO-PHOTON STIMULATION FOR READ-AND-WRITE NEURAL ACTIVITY

Understanding how neuronal circuits process information requires experimental approaches that can read neural activity and write precise perturbations into defined populations of neurons. Optical methods based on multiphoton excitation have become central to this effort, enabling minimally invasive, cell-resolved interrogation of neural circuits across space and time. However, all-optical experiments remain technically challenging, requiring careful integration of activity reporters, optogenetic actuators, and imaging and stimulation strategies that avoid cross-talk while preserving spatiotemporal precision.
Here, we aim to build an integrated optical platform for simultaneous recording and manipulation of neuronal activity with single-cell resolution. Neural activity will be read out via calcium imaging using genetically encoded calcium indicators, enabling extraction of functional connectivity patterns from population dynamics. For optical writing, neuronal firing will be controlled using two-photon optogenetic stimulation of the fast opsin ChroME2f. Our plan is to combine an inverted selective plane illumination microscope with nonlinear excitation (920nm) for fast volumetric calcium imaging, with a random-access stimulation module operating at 1030nm. The stimulation pathway will be based on a DMD, allowing flexible spatial targeting at kilohertz-rate of individual neurons. This architecture should enable independent optimization of read and write modalities while minimizing phototoxicity and unwanted opsin activation. By selectively activating neurons and monitoring the resulting calcium dynamics across the field of view, this approach will allow systematic mapping and manipulation of functional connectivity within neural circuits. The versatility of the platform makes it applicable to a wide range of experimental questions requiring all-optical interrogation of circuit dynamics and plasticity.