Dataset Information

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

ABSTRACT: Optical clearing techniques and light sheet microscopy have transformed fluorescent imaging of rodent brains, and have provided a crucial alternative to traditional confocal or bright field techniques for thin sections. However, clearing and labeling human brain tissue through all cortical layers and significant portions of a cortical area, has so far remained extremely challenging, especially for formalin fixed adult cortical tissue. Here, we present MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling approach for optically cleared human cortex samples, which can be applied to large (up to 5?mm thick) formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dye protocols in combination with new refractive index matching solutions allows deep volume imaging. This greatly reduces time and cost of imaging cytoarchitecture in thick samples and enables classification of cytoarchitectonic layers over the full cortical depth. We demonstrate application of MASH to large archival samples of human visual areas, characterizing cortical architecture in 3D from the scale of cortical areas to that of single cells. In combination with scalable light sheet imaging and data analysis, MASH could open the door to investigation of large human cortical systems at cellular resolution and in the context of their complex 3-dimensional geometry.

SUBMITTER: Hildebrand S

PROVIDER: S-EPMC6659684 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

Hildebrand Sven S Schueth Anna A Herrler Andreas A Galuske Ralf R Roebroeck Alard A

Scientific reports 20190726 1

Optical clearing techniques and light sheet microscopy have transformed fluorescent imaging of rodent brains, and have provided a crucial alternative to traditional confocal or bright field techniques for thin sections. However, clearing and labeling human brain tissue through all cortical layers and significant portions of a cortical area, has so far remained extremely challenging, especially for formalin fixed adult cortical tissue. Here, we present MASH (Multiscale Architectonic Staining of H ...[more]

PMID: 31350519

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Project description:Multi-modal imaging, by light-microscopy (LM) and Magnetic Resonance Imaging (MRI), holds promise for examining the brain across various resolutions and scales. While MRI acquires images in three dimensions, acquisition of intact whole-brain by LM requires a process of tissue clearing that renders the brain transparent. Removal of lipids (delipidation) is a critical step in the tissue clearing process, and was previsouly suggested to be the cause for absence of MRI contrast in cleared brains. Yet, the association between MRI contrast, delipidation and the different clearing techniques is debatable. Here, we provide datasets concerning lipid-content in cleared brain tissues obtained by various approaches. Fixed mouse and rat brains were cleared by CLARITY, Scale, uDISCO and ECi clearing techniques. Lipid-content was assessed at various intermediate steps of the different clearing methods, as well as at the end of the processes. Methods employed included whole brain MRI acquisition, Oil Red O (ORO)- and carbocyanine DiI-staining of cryosections, and DiI-washout assay from brain slices. MRI contrast-to-noise ratio, staining intensities and integrity of tissue were systematically analyzed. We demonstrate that lipid electrophoresis, an essential step of the CLARITY approach, engenders progressive reduction in MRI contrast in non-cleared (PFA-fixed) control brains, as well as strongly reduces contrast from uDISCO and ECi-cleared brains. ORO minimally stained CLARITY-cleared brains, however efficiently labelled uDISCO and ECi-cleared brains. Conversely, and in contrast to ORO-staining, DiI equally stained control, CLARITY, ECi and uDISCO-cleared brains. Both ORO- and DiI-staining demonstrated impairment in brain tissue integrity following CLARITY, but less so in uDISCO and ECi brains. DiI-washout assay demonstrated that each of the solvents employed along the process of uDISCO and ECi are highly delipidating, as well as the SDS-electrophoresis employed during CLARITY clearing. However, Scale treatment preserved most of the DiI dye. These data emphasize the variability in lipid assessment of cleared tissues by common techniques, and may help to resolve the contribution of lipids in brain MRI contrast.

Project description:In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10-12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

Dataset Information

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

Publications

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

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