Integrated platform for multi-scale molecular imaging and phenotyping of the human brain

Abstract

A detailed understanding of the anatomical and molecular architectures of brain cells and their brain-wide organization is essential for interrogating human brain function and dysfunction. Extensive efforts have been made toward mapping brain cells through various lenses, and have established invaluable databases yielding new insights. However, we still lack technologies that allow us to capture multi-scale multi-omic properties of individual brain cells in a holistic and scalable manner. To address this challenge, we developed a fully integrated technology platform for scalable three-dimensional (3D) mapping of human brain cells at subcellular resolution by simultaneously extracting brain-wide structure and high-dimensional features (e.g., spatial, molecular, morphological, and connectivity information) of individual cells from the same brain. We accomplished this by seamlessly integrating new chemical, mechanical, and computational tools to enable highly multiplexed multi-scale 3D proteomic imaging of human brain tissues with minimal information loss. We developed a novel microtome, termed MEGAtome, that enables ultra-precision slicing of a whole-mount intact human brain hemisphere and large arrays of animal organs with minimal loss of intra- and inter-slice information. To preserve structural and molecular information within intact human brain slabs and to enable highly multiplexed multiscale imaging, we developed a tissue-gel technology, termed mELAST, that transforms human brain tissues into an elastic, thermochemically stable, and reversibly expandable tissue-hydrogel. To reconstruct the intact brain to preserve the 3D connectivity information of neural fibers, we developed a semi-automatic tissue reconstruction computational pipeline termed HuBRIS. We demonstrated the utility and scalability of the technology platform by processing whole human brain hemispheres, investigating Alzheimer Disease (AD) pathology, and demonstrating the feasibility of projectome mapping.

IMPASTO: Multiplexed cyclic imaging without signal removalviaself-supervised neural unmixing

“…Tissue-clearing techniques enable the volumetric imaging of thick tissues without thin-sectioning 20,22,[38][39][40][44][45][46][47][48] , while tissue expansion techniques enable the super-resolution imaging of proteins in thick tissue slices 21,31,[49][50][51][52][53][54][55][56][57][58][59][60] . Repeated staining, imaging, and antibody removal have been used in the molecular imaging of cleared 20,21 or expanded tissues 5,55,61,62 . In such cyclic imaging, the signal removal step could be replaced with IMPASTO's neural unmixing algorithm.…”

“…Existing cyclic imaging techniques can be divided into three categories based on the signal removal mechanisms: photobleaching-based, chemical-inactivation-based, and antibody-strippingbased methods. In photobleaching-based methods, the fluorophores of antibodies are bleached by directly illuminating the target specimens with a high intensity light 5,6 . In chemical-inactivation-based methods, the fluorophores of antibodies are deactivated by chemical treatment [7][8][9][10][11] .…”

Efficient image analysis for large-scale next generation histopathology using pAPRica

“…This approach is usually carried out in small tissue sections of a few micrometer thickness, thus introducing a sampling bias in the assessment of biological and pathological processes. Next generation histopathology methods propose to analyze large organ parts at high resolution thanks to the use of new tissue preparation protocols and large-scale volumetric microscopy techniques [1][2][3][4] . Their wider adoption in research and in the clinic, however, is currently limited by the lack of appropriate methods to efficiently process and analyze the terabyte-sized datasets they generate.…”

Microscale visualization of cellular features in adult macaque visual cortex

“…Expansion microscopy (ExM) facilitates nanoscale imaging of biological structures with diffraction-limited microscopy in a wide variety of tissue preparations. [1][2][3][4][5][6][7][8][9][10][11][12][13] In brain tissue, ExM enables discrete visualization of hydrophilic biomolecules such as proteins, DNA, and mRNA, which are densely packed within and around hydrophobic myelin sheaths, by physically separating hydrophobic and hydrophilic tissue regions through isotropic expansion 3,7,12,13 . Detailed analyses of cellular features across large samples of brain tissue become feasible when ExM is combined with light sheet fluorescence microscopy (LSFM), which accelerates acquisition of high resolution image data in cleared tissues.…”

“…Detailed analyses of cellular features across large samples of brain tissue become feasible when ExM is combined with light sheet fluorescence microscopy (LSFM), which accelerates acquisition of high resolution image data in cleared tissues. [14][15][16][17] This combinatorial technique is particularly useful when investigating neuroanatomy and connectivity in large, gyrencephalic nonhuman primate and human brains, 5,[11][12][13][14] where prior studies are limited to sparsely labeled neural populations or subsets of cellular features within neural circuits. 18…”