A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing

Murakami, Tatsuya C.; Mano, Tomoyuki; Saikawa, Shu; Horiguchi, Shuhei A.; Shigeta, Daichi; Baba, Kousuke; Sekiya, Hiroshi; Shimizu, Yoshihiro; Tanaka, Kenji F.; Kanazawa, Hiroshi; Iino, Masamitsu; Mochizuki, Hideki; Tainaka, Kazuki; Ueda, Hiroki R.

doi:10.1038/s41593-018-0109-1

Cited by 237 publications

(273 citation statements)

References 63 publications

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“…[22] It should be noted that the current low-number counting results were obtained using a thy1-GFP-M transgenic mouse, in which GFP signal is expressed by less than 10% of all motor axons, retinal ganglion cells, lumbar dorsal root ganglions, and cortex. [22] It should be noted that the current low-number counting results were obtained using a thy1-GFP-M transgenic mouse, in which GFP signal is expressed by less than 10% of all motor axons, retinal ganglion cells, lumbar dorsal root ganglions, and cortex.…”

Section: Whole-brain Visualization and Segmentationmentioning

confidence: 84%

Fast, 3D Isotropic Imaging of Whole Mouse Brain Using Multiangle‐Resolved Subvoxel SPIM

Nie

Liu

et al. 2019

Advanced Science

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challenges in neuroscience. To unravel the various neuronal profiles of different physiological functions in the whole brain, 3D high-resolution (HR) imaging is required over a mesoscale sized volume. [1] However, creating such a large-scale brain dataset has posed a big challenge for current 3D optical microscopy methods, all of which show relatively small optical throughputs. [2,3] Furthermore, light scattering and attenuation are outstanding issues for the turbid tissues that limit the extraction of signals from deep brain. To address these issues, 3D tile stitching combined with brain sectioning has been a popular strategy for obtaining mammalian brain atlases, which can be a meaningful platform for mapping neuronal populations, activities, or connections over the entire brain. [4] For example, sequential two-photon tomography (STPT) can 3D image the brain at subcellular high resolution, [5,6] but at the cost of long acquisition times of up to several days and a highmaintenance system setup. The advent of light-sheet fluorescence microscopy [7] (LSFM) in conjunction with tissue-clearing [8] eliminates the need for complicated mechanical slicing of samples by instead applying nondestructive optical sectioning. Although LSFM still needs repetitiveThe recent integration of light-sheet microscopy and tissue-clearing has facilitated an important alternative to conventional histological imaging approaches. However, the in toto cellular mapping of neural circuits throughout an intact mouse brain remains highly challenging, requiring complicated mechanical stitching, and suffering from anisotropic resolution insufficient for highquality reconstruction in 3D. Here, the use of a multiangle-resolved subvoxel selective plane illumination microscope (Mars-SPIM) is proposed to achieve high-throughput imaging of whole mouse brain at isotropic cellular resolution. This light-sheet imaging technique can computationally improve the spatial resolution over six times under a large field of view, eliminating the use of slow tile stitching. Furthermore, it can recover complete structural information of the sample from images subject to thick-tissue scattering/ attenuation. With Mars-SPIM, a digital atlas of a cleared whole mouse brain (≈7 mm × 9.5 mm × 5 mm) can readily be obtained with an isotropic resolution of ≈2 µm (1 µm voxel) and a short acquisition time of 30 min. It provides an efficient way to implement system-level cellular analysis, such as the mapping of different neuron populations and tracing of long-distance neural projections over the entire brain. Mars-SPIM is thus well suited for high-throughput cellprofiling phenotyping of brain and other mammalian organs.

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Section: Whole-brain Visualization and Segmentationmentioning

confidence: 84%

Fast, 3D Isotropic Imaging of Whole Mouse Brain Using Multiangle‐Resolved Subvoxel SPIM

Nie

Liu

et al. 2019

Advanced Science

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“…become available for regions outside of the neocortex and thalamus, which MISS can easily accommodate to obtain maps of additional cell types. Alternative approaches that directly quantify cell types using fluorescence imaging, in addition to being limited in terms of their spatial coverage, require sophisticated and expensive microscopy platforms that may be inaccessible to many researchers looking to understand how cell types they identify transcriptomically are distributed throughout the brain 1,3,4,7,8 .…”

Section: Comparison To Cell Type Quantification Methodsmentioning

confidence: 99%

“…Characterizing whole-brain distributions of different neural cell types, especially different subtypes of GABAergic (inhibitory), glutamatergic (excitatory) cells, is a topic of keen interest in modern neuroanatomy with many applications to both basic and clinical neuroscience research. Advances in molecular methods for quantifying gene expression and data analytic cell clustering techniques based on morphologic or genetic profiles [1][2][3][4][5][6][7][8] are beginning to enable the mapping of meso-and-microscale neuronal and non-neuronal cell type architecture at a whole-brain scale. Pioneering work mapping single-cell RNA sequencing (scRNAseq) data from aquatic flatworms and zebrafish onto in situ hybridization (ISH) expression provides a plausible route to mammalian cell-type mapping in the nervous system 9,10 .…”

Section: Introductionmentioning

confidence: 99%

Matrix Inversion and Subset Selection (MISS): A novel pipeline for mapping of diverse cell types across the murine brain

Mezias

Torok

Maia

et al. 2019

Preprint

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The advent of increasingly sophisticated imaging platforms has allowed for the visualization of the murine nervous system at single-cell resolution. However, current cell counting methods compromise on either the ability to map a large number of different cell types, or spatial coverage, and therefore whole-brain quantification of finely resolved neural cell subtypes remains elusive. Here we present a comprehensive and novel computational pipeline called Matrix Inversion and Subset Selection (MISS) that aims to fill this gap in knowledge and has the ability to infer counts of diverse collections of neural cell types at sub-millimeter resolution using a combination of single-cell RNAseq and in situ hybridization datasets. We rigorously demonstrate the accuracy of MISS against literature expectations, and in so doing, provide the first verified maps for twenty-five distinct neuronal and nonneuronal cell types across the whole mouse brain. The resulting comprehensive cell type maps are uniquely suited to address important open questions in neuroscience. As a demonstration, we utilize our inferred maps to quantitatively establish that adult cell type distributions reflect the ontological splits between brain regions during neural development, indicating that the developmental history of brain regions informs their final cell-type composition. Although this link has been frequently surmised by neuroscientists, its quantitative verification across the entire brain has not previously been demonstrated. Together, our results suggest that the MISS pipeline can be used to generate accurate spatial quantification of diverse cell types without the direct imaging of known type-specific markers, as has been done previously. The entire MISS pipeline and outputs will be made open source in order to catalyze future neuroscientific advances across disciplines.

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“…30 (b) Aqueous reagent-based clearing methods, including Scale, 31 ClearT, 32 SeeDB, 33 CUBIC series. 25,34,35 (c) Hydrogel-based clearing methods, including CLARITY, 24 PACT. 21 Three major criteria to evaluate a clearing method include transparency outcome, fluorescent preservation and applicability of tissues.…”

Section: Introductionmentioning

confidence: 99%

3‐dimensional visualization of implant‐tissue interface with the polyethylene glycol associated solvent system tissue clearing method

Men

Jing

et al. 2019

Cell Proliferation

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Objectives: Dental implants are major treatment options for restoring teeth loss.Biological processes at the implant-tissue interface are critical for implant osseointegration. Superior mechanical properties of the implant constitute a major challenge for traditional histological techniques. It is imperative to develop new technique to investigate the implant-tissue interface. Materials and methods:Our laboratory developed the polyethylene glycol (PEG)-associated solvent system (PEGASOS) tissue clearing method. By immersing samples into various chemical substances, bones and teeth could be turned to transparent with intact internal structures and endogenous fluorescence being preserved. We combined the PEGASOS tissue clearing method with transgenic mouse line and other labelling technique to investigate the angiogenesis and osteogenesis processes occurring at the implant-bone interface.Results: Clearing treatment turned tissue highly transparent and implant could be directly visualized without sectioning. Implant, soft/hard tissues and fluorescent labels were simultaneously imaged in decalcified or non-decalcified mouse mandible samples without disturbing their interfaces. Multi-channel 3-dimensional image stacks at high resolution were acquired and quantified. The processes of angiogenesis and osteogenesis surrounding titanium or stainless steel implants were investigated. Conclusions:Both titanium and stainless steel implants support angiogenesis at comparable levels. Successful osseointegration and calcium precipitation occurred only surrounding titanium, but not stainless steel implants. PEGASOS tissue clearing method provides a novel approach for investigating the interface between implants and hard tissue.

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A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing

Cited by 237 publications

References 63 publications

Fast, 3D Isotropic Imaging of Whole Mouse Brain Using Multiangle‐Resolved Subvoxel SPIM

Fast, 3D Isotropic Imaging of Whole Mouse Brain Using Multiangle‐Resolved Subvoxel SPIM

Matrix Inversion and Subset Selection (MISS): A novel pipeline for mapping of diverse cell types across the murine brain

3‐dimensional visualization of implant‐tissue interface with the polyethylene glycol associated solvent system tissue clearing method

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