In-vivo neuroimaging using adaptive optics micrcoscopy – INOVAO

The main technological brick of the project consists in a new, extended-source Shack-Hartmann wavefront sensor, which does not require the use of a «guide star« that generates a unique wavefront in the volume of a sample. Such a guide star is currently either not possible in routine biological use or responsible for high instrumental complexity. This sensing approach enables a simplified implementation of adaptive optics in microscopy, in particular in the case of optical sectioning microscopes. Moreover, since it is a direct wavefront sensing method, it provides optimal performance of the adaptive optics process, as previously demonstrated in the state of the art.

During the first part of the project, we developed a new, extended-source Shack-Hartmann wavefront sensor, optimized for use in fluorescence microscopy, and demonstrated its metrology performance. A light-sheet microscope integrating an adaptive optics loop based on the previous sensor has been developed. It enabled to demonstrate a significant improvement of the quality of images of the live drosophila brain, without clarification, at depths of few tens of microns, by correcting sample and instrument induced aberrations. This new adaptive optics approach represents an excellent compromise between performance and instrumental simplicity.

The INOVAO project demonstrated the feasibility of a new adaptive optics approach providing significantly simplified implementation when compared to current approaches in the field of microscopy, in particular in neuroscience. The project is a first step toward commercial exploitation of adaptive optics modules and/or systems for microscopy, in particular regarding light-sheet and 2-photon microscopes.

Publications:
A. Hubert, F. Harms, R. Juvénal, P. Treimany, X. Levecq, V. Loriette, G. Farkouh, F. Rouyer, and A. Fragola, Adaptive optics light-sheet microscopy based on direct wavefront sensing without any guide star , Optics Letters 44 (10), 2514-2517 (2019)

Patents:
French patent Application FR1901011 - Dispositifs d’analyse de front d’onde et systèmes d’imagerie microscopique comprenant de tels dispositifs. filed: 01/02/2019

Understanding brain function is of key medical & biological importance, when targeting better understanding of the mechanisms underlying brain pathologies, such as Alzheimer’s disease, multiple sclerosis or absence epilepsy. This objective is however still a challenge, since brain is a complex and dynamic biological system, containing a huge number of neurons, with diverse morphology and connectivity, exhibiting distinct activity in the course of behaviors. Deciphering the functional organization of neuronal networks thus requires studying neuronal activity in large networks, with high temporal and spatial resolution, as well as limited invasiveness. To this aim, optical imaging approaches, combined with genetic tools, now offer increasing capabilities. Indeed, neuroimaging now benefits from efforts that have been made in parallel in biochemistry and in microscopy, providing genetic encoding methods to tag specific cell types through light-activated protein-encoded reporters, and providing microscopy setups capable of sub-cellular structural and functional non-invasive imaging in animal models such as mice or fruit flies. Among microscopy techniques, Two-Photon Excitation Fluorescence Microscopy (TPEF) and Light-Sheet Fluorescence Microscopy (LSFM) are particularly adapted to neuroimaging. The two methods currently achieve high-resolution (<1µm), high-sensitivity fluorescence imaging in intact neural tissue, over large field-of-view (typically 400x400µm), in depth (>200µm), measuring spatial distributions and dynamics of tagged proteins in-vivo.
Despite impressive results, both techniques currently suffer from several limitations:
• the weak fluorescence variations induced by neuronal activity (5 to 10% of relative variation of single action potentials evokes calcium transients)
• the limited depth penetration (typically up to a maximum of 300 to 500 µm)
• the optical resolution, which still needs to be increased.
One major cause of the loss of signal, in particular in depth, arise from optical aberrations due to inhomogeneities of biological tissue strongly distorting the phase of optical waves, i.e. the wavefront of the light of interest. In the recent years, adaptive optics has shown its ability to increase signal, resolution and imaging depth in microscopy. This is achieved through 1- wavefront sensing and 2 -correction using optical wavefront modulators like deformable mirrors.
In order to allow for improved functional imaging in neuroscience, the InovAO project will develop or achieve the following:
• an innovative adaptive optics approach based on direct wavefront sensing, optimized for sectioning microscopy, using a new wavefront sensor. This approach will significantly extend the corrected field-of-view, typically by a factor of 3.
• adaptive optics-TPEF & adaptive optics-LSFM demonstrator microscopes, integrating the previous AO approach, with increased spatio-temporal imaging capabilities
• biological in-vivo studies validating and quantifying the performance of the device for functional neuroimaging in mouse and drosophila brains, at the cellular level and on extended cells networks.
The new technique will overcome current AO limitations and represents the first step toward future commercial devices.