Enhanced neuroimaging with a calcium sensor in ex-vivo Drosophila melanogaster brains using closed-loop adaptive optics light-sheet fluorescence microscopy.

Significance: Adaptive optics (AO) has been implemented on several microscopy setups and has proven its ability to increase both signal and resolution. However, reported configurations are not suited for fast imaging of live samples or are based on an invasive or complex implementation method. Aim: Provide a fast aberration correction method with an easy to implement AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced imaging of live samples. Approach: Development of an AO add-on module for LSFM based on direct wavefront sensing without requiring a guide star using an extended-scene Shack-Hartmann wavefront sensor. The enhanced setup uses a two-color sample labeling strategy to optimize the photon budget. Results: Fast AO correction of in-depth aberrations in an ex-vivo adult Drosophila brain enables doubling the contrast when imaging with either cell reporters or calcium sensors for functional imaging. We quantify the gain in terms of image quality on different functional domains of sleep neurons in the Drosophila brain at various depths and discuss the optimization of key parameters driving AO. Conclusion: We developed a compact AO module that can be integrated into most of the reported light-sheet microscopy setups, provides significant improvement of image quality and is compatible with fast imaging requirements such as calcium imaging.

Single-shot quantitative aberration and scattering length measurements in mouse brain tissues using an extended-source Shack-Hartmann wavefront sensor.

Deep fluorescence imaging in mammalian brain tissues remains challenging due to scattering and optical aberration-induced loss in signal and resolution. Correction of aberrations using adaptive optics (AO) requires their reliable measurement in the tissues. Here, we show that an extended-source Shack-Hartmann wavefront sensor (ESSH) allows quantitative aberration measurements through fixed brain slices with a thickness up to four times their scattering length. We demonstrate in particular that this wavefront measurement method based on image correlation is more robust to scattering compared to the standard centroid-based approach. Finally, we obtain a measurement of the tissue scattering length taking advantage of the geometry of a Shack-Hartmann sensor.