Illumination angle correction during image acquisition in light-sheet fluorescence microscopy using deep learning.

Light-sheet fluorescence microscopy (LSFM) is a high-speed imaging technique that provides optical sectioning with reduced photodamage. LSFM is routinely used in life sciences for live cell imaging and for capturing large volumes of cleared tissues. LSFM has a unique configuration, in which the illumination and detection paths are separated and perpendicular to each other. As such, the image quality, especially at high resolution, largely depends on the degree of overlap between the detection focal plane and the illuminating beam. However, spatial heterogeneity within the sample, curved specimen boundaries, and mismatch of refractive index between tissues and immersion media can refract the well-aligned illumination beam. This refraction can cause extensive blur and non-uniform image quality over the imaged field-of-view. To address these issues, we tested a deep learning-based approach to estimate the angular error of the illumination beam relative to the detection focal plane. The illumination beam was then corrected using a pair of galvo scanners, and the correction significantly improved the image quality across the entire field-of-view. The angular estimation was based on calculating the defocus level on a pixel level within the image using two defocused images. Overall, our study provides a framework that can correct the angle of the light-sheet and improve the overall image quality in high-resolution LSFM 3D image acquisition.