Meso-Aspheric based tunable light-sheet microscopy: reliable technique for precision imaging of neural and vascular network

Abstract

Meso-aspheric optics provide complex optical elements that enhance imaging techniques, particularly in high-resolution microscopy. In this work, we present the latest advancements in our tunable light-sheet microscope. By incorporating refractive optics, such as achromatic-aspheric and meso-aspheric lenses, alongside diffractive optics, we have enhanced optical performance, enabling high-resolution 2D and 3D imaging of the neural and vascular networks in biological tissues of varying sizes, as shown in Fig. 1-A. To prepare the specimens, we use an optimized clearing technique involving an organic clearing agent (DBE), specific wavelength light sources, and ultrasonic waves. The specimens are then fixed and secured in a clamp within a container filled with a clearing medium that matches the refractive index. The newly developed tunable light-sheet system generates an ultra-thin, uniform-intensity light sheet with an extended Rayleigh range, as shown in Fig. 1B–D. The system is equipped with three continuous Gaussian beam lasers (488 nm, 532 nm, and 660 nm) for fluorescence excitation. Additional components include optical filters, two custom-made light sheet generators for dual-sided illumination, computer-controlled vertical and horizontal stages, and specialized objectives that compensate for refractive index mismatches between air and the immersion medium. Image acquisition is carried out using an Andor Neo CMOS camera (2560 × 2160 pixels), followed by post-processing with NeuroDeblur deconvolution software and 3D reconstruction using AMIRA 2020-2. This tunable light-sheet microscope represents a significant advancement in 3D imaging, offering high resolution across all three axes for enhanced biological visualization.