Wavefront Engineering Microscopy

Presentation

Combined with optogenetic actuators and high-power amplified lasers, these optical strategies enable precise spatiotemporal control of the activity of single or multiple targeted neurons. This allowed manipulation of neural circuits across millimeter-sized volumes, with cellular spatial resolution and millisecond-range temporal precision in the mouse cortex, both in vitro and in vivo. Coupled with calcium imaging, we demonstrated all-optical read/write interfaces for simultaneously monitoring and manipulating neuronal activity, enabling fine-scale functional mapping of neural circuits. By integrating multiphoton holography with endoscopy, the group has demonstrated simultaneous photostimulation and fast functional imaging (up to 80 Hz) at cellular resolution in freely moving mice, targeting both cortical areas and the hippocampus. More recently, the group has shown that holographic light shaping combined with temporal focusing enables high-contrast, high-resolution in vitro and in vivo voltage imaging in densely labeled preparations, including mouse cortex and zebrafish larvae expressing GFP- and rhodopsin-based genetically encoded voltage indicators.
These research efforts are organized around four main domains:

1. Large-scale holographic optogenetics and voltage imaging
   We develop optical strategies for large-scale, two-photon multi-target photostimulation and functional (Ca²⁺ or voltage) imaging. These approaches enable precise, parallel illumination of large neuronal ensembles while maintaining high temporal and spatial resolution. Ultimately, this work aims to enable high-throughput, all-optical large-scale mapping of functional neuronal connectivity across extended and anatomically distributed brain regions in mice.
2. In-depth all-optical circuit manipulation with three-photon holographic microscopy
   We develop advanced three-photon holographic approaches for precise, cell-resolved optical stimulation and functional imaging of neuronal populations deep in living tissue. These methods support new experimental paradigms to probe the organization and dynamics of neural circuits across cortical layers.
3. All-optical manipulation of retinal circuits
   We develop all-optical read–write interfaces for the non-invasive interrogation of retinal circuits across multiple functional layers. These systems enable simultaneous control of retinal inputs to photoreceptors, manipulation of internal retinal processing circuits, and monitoring of neuronal responses at the level of retinal ganglion cells in mouse retina and non-human primate retinal explants.
4. All-optical manipulation in freely moving animals
   We have developed a new class of flexible two-photon microendoscopes (2P-FENDO) that enable near-cellular–resolution imaging and optogenetic control of neuronal activity in cortical layer of freely moving mice providing fast imaging and large fields of view of up to ~500 µm. Recent 2P-FENDO architectures enable in-depth imaging and targeted photostimulation in deep brain regions such as the hippocampus. Together, these instruments provide unique capabilities to monitor and perturb neural circuits in freely behaving animals, opening new avenues for studying the neural basis of behavior in naturalistic relevant conditions.