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Today, Light Sheet Fluorescence Microscopy (LSFM) makes it possible to image fluorescent samples through depths of several hundreds of microns. However, LSFM also suffers from scattering, absorption and optical aberrations. Spatial variations in the refractive index inside the samples cause major changes to the light path resulting in loss of signal and contrast in the deepest regions, thus impairing in-depth imaging capability. These effects are particularly marked when inhomogeneous, complex biological samples are under study. Recently, chemical treatments have been developed to render a sample transparent by homogenizing its refractive index (RI), consequently enabling a reduction of scattering phenomena and a simplification of optical aberration patterns. One drawback of these methods is that the resulting RI of cleared samples does not match the working RI medium generally used for LSFM lenses. This RI mismatch leads to the presence of low-order aberrations and therefore to a significant degradation of image quality. In this paper, we introduce an original optical-chemical combined method based on an adaptive SPIM and a water-based clearing protocol enabling compensation for aberrations arising from RI mismatches induced by optical clearing methods and acquisition of high-resolution in-depth images of optically cleared complex thick samples such as Multi-Cellular Tumour Spheroids.

High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM