Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues

Abstract

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

Dissecting Neuronal Activation on a Brain-Wide Scale With Immediate Early Genes

“…While transgenic strategies are currently not applicable to nonhuman primates (NHP), viral approaches like RAM could be, in principle, switched to map-activated neurons in different animals (Sørensen et al, 2016). Indeed, large-scale clearing and imaging methods have been demonstrated in small NHP like the marmoset (Seiriki et al, 2017;Susaki et al, 2020). When coupled with standard neuroimaging like functional magnetic resonance imaging (fMRI) or positron-emission tomography (PET), single-cell activation mapping in NHP could provide a unique framework to better understand the relationship between neuronal activity and imaging data.…”

“…Anyhow, whatever optical improvement must rely on a good optical clearing, which is an essential prerequisite for the use of high-resolution LSM. The group of Hiroki Ueda has pioneered IEG mapping at subcellular resolution, demonstrating the potential of this approach to quantify neuronal activation across the entire murine brains without excluding any areas (Murakami et al, 2018;Matsumoto et al, 2019;Susaki et al, 2020).…”

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

“…This type of tissue is the most widely studied using tissue clearing methods ( Kim et al., 2018 ; Wan et al., 2018 ). In principle, it is essential for any method that aims at clearing neural tissue to perform a prior delipidation step to remove the myelin sheets that enclose neuronal axons ( Susaki et al., 2020 ), as their high lipidic content causes a large amount of scattering. An approach combining CLARITY with double photon microscopy enabled visualization of gray matter in the intact brain ( Chang et al., 2017 ).…”

“…CUBIC was proposed as a new brain clearing method that obviates electrophoresis, in contrast to CLARITY ( Susaki et al., 2014 ). CUBIC's clearing capability of neural tissue has been quantitatively proved by means of a small-angle X-ray scattering analysis (SAXS), which showed that CUBIC reagent 1 is capable of effectively removing the myelin sheet and, therefore, reducing scattering ( Susaki et al., 2020 ). Chicken embryo brains have been successfully cleared with an optimized CUBIC method ( Gómez-Gaviro et al., 2017 ).…”

“…The authors demonstrated the electrolyte-hydrogel behavior of fixed, delipidated tissues, what supports the application of the Lorentz-Lorenz equation (which was stated in 1880 for polymers) to tissues, as mentioned in the introduction ( Ueda et al., 2020 ). They used this resemblance to optimize electrostatic interactions between the dye or immunolabel and the gel, and improve their penetration by regulating pH, salt concentration, temperature, and other environmental and chemical conditions ( Susaki et al., 2020 ).…”