Sensitive label-free imaging of brain samples using FxClear-based tissue clearing technique

Abstract

Summary Optical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.

MAX: a simple, affordable, and rapid tissue clearing reagent for 3D imaging of wide variety of biological specimens

“…Previously, we demonstrated that label-free imaging with selective-plane illumination microscopy (SPIM) microscopy is applicable to whole-organ imaging in combination with a tissue-clearing procedure 24 . The source of label-free signals is multiple organic materials including lipids.…”

“…The efficiency of these processes is strongly associated with hydrophobicity/hydrophilicity of biological specimens. For instance, tissue transparency is differentially affected by lipid content and the average RI value 17 , 24 – 27 . In lipid-rich organs, de-lipidation is essential to achieve a substantial degree of transparency.…”