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.

Studying the trafficking of labeled trodusquemine and its application as nerve marker for light‐sheet and expansion microscopy

“…The whole slice acquisition was performed using a custom‐made, dual‐view, inverted light sheet fluorescence microscope with two 12×, NA 0.53, WD 10 mm excitation and emission objectives which are tilted at 45° relative to the sample plane. The whole system is controlled with a custom data acquisition and control software specifically developed for this setup, as reported in Pesce et al 35 …”

Efficient 3D light-sheet imaging of very large-scale optically cleared human brain and prostate tissue samples

“…Various labs have developed optical clearing protocols, such as CLARITY 6 , 8 , iDISCO 9 , 10 and CUBIC 12 , which have mainly been applied to render mouse brains transparent, in order to understand both the brain’s structure and pathology 7 . However, the application of cleared tissue light-sheet imaging to large archival (i.e., fixed with aldehyde fixatives) adult human brain samples, in particular, has been a major challenge because of the sample size and the difficulties of applying clearing, labelling and imaging throughout large volumes of myelin-rich tissue 13 .…”

Editorial: The human brain multiscale imaging challenge

“…Such specimens present specific challenges that need to be solved in comparison to animal models: massive dimension of the specimen (up to several cm 3 ), geometry, variability of post-mortem fixation conditions and storage, presence of blood inside the vasculature, autofluorescence signals from lipofuscin-type pigments, and consistency of cellular labeling. In addition, alteration of antigens, due to fixation and storage conditions, may prevent reliable immunostaining (Weiss et al, 2021 ; Pesce et al, 2022 ). Various optical technologies have started to address human brain reconstruction in combination with advanced staining methods or relying on label-free detections (Axer et al, 2016 ; Wang et al, 2018 ; Menzel et al, 2020 ; Costantini et al, 2021a ) but much remains to be done.…”