A multimodal imaging and analysis pipeline for creating a cellular census of the human cerebral cortex

Abstract

Cells are not uniformly distributed in the human cerebral cortex. Rather, they are arranged in a regional and laminar fashion that span a range of scales. Here we demonstrate an innovative imaging and analysis pipeline to construct a reliable cell census across the human cerebral cortex. Magnetic resonance imaging (MRI) is used to establish a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with both traditional immunohistochemistry, to provide a stereological gold-standard, and with a custom-made inverted confocal light-sheet fluorescence microscope (LSFM) for 3D imaging at cellular resolution. Finally, mesoscale optical coherence tomography (OCT) enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system.

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy

“…Human brain samples were preliminarily treated for fluorescence microscopy following the label-free MAGIC preparation technique, developed by Costantini (Costantini et al (2021)). This method originates from the evidence that glycerol removal from fixed and embedded brain tissue leads to a specific increase in myelin autofluorescence and thus to an enhanced signal-to-background ratio of myelinated axons.…”

“…The point spread function (PSF) of the TPFM system was previously characterized in (Costantini et al (2021)), by imaging 100 nm beads (FluoSpheresTM carboxylate-modified microspheres, yellow-green fluorescent, Thermo Fisher Scientific, US) embedded at a 1:1000 concentration in a PBS gel reproducing the refractive index of the immersion medium employed for the brain slice imaging. The average shape of the PSF was estimated using the Huygens PSF distiller tool (version 19.04, Scientific Volume Imaging, NL), resulting in a measured FWHM of (0.692, 0.692, 2.612) μm along the x, y, and z axes, respectively.…”

“…Therefore, the development of a gold standard method capable of providing reliable multiscale ground-truth datasets for the comprehensive validation of dMRI-based connectivity information is of paramount importance. This has motivated several research groups to apply dMRI to post mortem histological tissue sections in combination with a range of optical modalities, namely 3D polarized light imaging (3D-PLI) (Axer et al (2011, 2016)), 3D polarized-sensitive optical coherence tomography (3D-PSOCT) (Wang et al (2018); Jones et al (2020)), 2D and 3D confocal scanning fluorescence microscopy (Budde and Frank (2012); Khan et al (2015); Schilling et al (2018)) and 3D light-sheet fluorescence microscopy (LSFM) (Morawski et al (2018)), also combining OCT and LSFM (Costantini et al (2021)), with the aim to identify suitable approaches for validating dMRI-based fiber tractography, and unveiling its potential limitations in brain regions with challenging fiber compositions and geometrical configurations. 3D-PLI and 3D-PSOCT exploit the inherent birefringence of brain tissue, analyzing the change in polarization of light transmitted through unstained samples in order to detect fiber structures and estimate their 3D orientation.…”

“…3D-PLI and 3D-PSOCT exploit the inherent birefringence of brain tissue, analyzing the change in polarization of light transmitted through unstained samples in order to detect fiber structures and estimate their 3D orientation. On the other hand, fluorescence microscopy generally relies on tissue clearing methods, such as CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) (Chung and Deisseroth (2013); Costantini et al (2015)), or SWITCH (System-Wide control of Interaction Time and kinetics of CHemicals) (Murray et al (2015); Costantini et al (2021); Pesce et al (2022)) to render samples transparent, and histological stains for generating contrast between fibers and other tissue components. Furthermore, despite offering a superior spatial resolution with respect to polarimetry-based imaging modalities, which enables the targeting of finer microstructural details, in fluorescence microscopy the evaluation of fiber orientations is subject to the application of dedicated image processing techniques, namely Fourier analysis (Budde et al (2011); Choe et al (2012)) and structure tensor analysis (Budde and Frank (2012); Khan et al (2015); Schilling et al (2018)).…”

“…However, their orientation analysis was limited to the image plane due to the marked anisotropic resolution of the employed light-sheet microscope and, moreover, it relied on the availability of manual segmentations produced by expert neuroanatomists. In this work, we combined the sub-micron resolution and deep imaging capabilities offered by two-photon fluorescence microscopy (TPFM), with the myelin autofluorescence enhancement achieved by a pioneering label-free preparation protocol named MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement) (Costantini et al (2021)), for acquiring high-resolution mesoscale reconstructions of the human brain fiber organization. Large volumetric brain tissue images were analyzed by means of a novel image processing pipeline built around a 3D implementation of the Frangi filter, able to further enhance myelinated fiber structures of varying diameters and produce 3D orientation maps preserving the native resolution of the TPFM system.…”