3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy

Abstract

The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH - H2O2 - antigen Retrieval - TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.

Comparison of Different Tissue Clearing Methods for Three-Dimensional Reconstruction of Human Brain Cellular Anatomy Using Advanced Imaging Techniques

“…We also tested the SWITCH and SHIELD protocols that both yielded uniform clearing of the white and gray matter (Figures 1D,E). These clearing protocols optimized for the human brain showed an efficient clearing process in the white and gray matter of the tissue, demonstrating the compatibility of SHIELD protocol with the TDE refractive index matching medium (Costantini et al, 2015;Pesce et al, 2021b). In parallel, we assessed ExM as a clearing protocol variant (Chen et al, 2015;Gao et al, 2017), to investigate 100 µm-thick slices (Figure 1A and Supplementary Figure 1A).…”

“…The SWITCH protocol was performed following the protocol of Pesce et al (2021b) . The samples were incubated at 4°C for 24 h in 20 ml of SWITCH-Off solution, consisting of 50% PBS titrated to pH 3 using HCl, 25% 0.1 M HCl, 25% 0.1 M potassium hydrogen phthalate (KHP) and 4% glutaraldehyde.…”

“…To reduce autofluorescence in each clearing method, we tested two different treatments alone or in combinations ( Pesce et al, 2021b ). Here, we report the optimized protocols for each clearing method ( Supplementary Figure 1A ).…”

“…Finally, the samples were equilibrated in PBS for at least 1 h at RT. For the SWITCH-processed slice, the decolorization protocol was performed according to the SHORT protocol ( Pesce et al, 2021b ). In particular, after performing SWITCH protocols, samples were treated with H 2 O 2 and AR except that the clearing process was performed first.…”

“…Different strategies to increase the pore size of fixed specimens ( Chung et al, 2013 ; Gleave et al, 2013 ; Lai et al, 2018 ; Wassie et al, 2019 ), as well as reducing non-specific antibody-samples interactions ( Hama et al, 2015 ; Murray et al, 2015 ), and also increasing active transports by electrophoresis ( Lee et al, 2016 ; Wang et al, 2018 ) can be used to reduce tissue density and improve probe penetration speed into large samples. Another important challenge in tissue staining is reaching a homogeneous antibody labeling against high-density epitopes ( Chung et al, 2013 ; Ueda et al, 2020b ; Pesce et al, 2021b ). The best candidate in terms of distribution density in neuronal somata is the neuronal nuclear antigen (NeuN), which is widely used for quantitative neuronal morphometric studies of human brain tissue ( Gittins and Harrison, 2004 ).…”