Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

Yang, Baohua; Treweek, Jennifer B.; Kulkarni, Rajan P.; Deverman, Benjamin E.; Chen, Chun Kan; Lubeck, Eric; Shah, Sheel; Cai, Long; Gradinaru, Viviana

doi:10.1016/j.cell.2014.07.017

Cited by 808 publications

(1,119 citation statements)

References 38 publications

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“…Many modern tissue-clearing approaches are designed to preserve the protein content of the sample while reducing scattering and autofluorescence background by extracting lipids and matching refractive index (16)(17)(18)(19)(20)(21)(22)(23). For example, embedding and cross-linking tissues to hydrogels provides a powerful approach to tissue clearing, minimizing sample distortion during lipid removal and index matching while maintaining the protein content of the sample (19,20). These approaches have also been made compatible with RNA FISH by stabilizing RNA molecules, for example through crosslinking of RNAs to proteins, without removing the protein content of the cell (20,24).…”

Section: Significancementioning

confidence: 99%

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Moffitt

Hao

Bambah-Mukku

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

233

249

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Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, backgroundfrom off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.tissue clearing | fluorescence in situ hybridization | multiplexed imaging | single-cell transcriptomics | brain S ingle-molecule FISH (smFISH) is a powerful technique that allows the direct imaging of individual RNA molecules within single cells (1, 2). In this approach, RNAs are labeled via the hybridization of fluorescently labeled oligonucleotide probes, producing bright fluorescent spots for single RNA molecules, which reveal both the abundance and the spatial distribution of these RNAs inside cells (1, 2). The ability of smFISH to image gene expression at the single-cell level in both cell culture and tissue has led to exciting advances in our understanding of the natural noise in gene expression and its role in cellular response (3, 4), the intracellular spatial organization of RNAs and its role in posttranscriptional regulation (5, 6), and the spatial variation in gene expression within complex tissues and its role in the molecular definition of cell types and tissue functions (6, 7).To extend the benefits of this technique to systems-level questions and high-throughput gene-expression profiling, approaches to increase the multiplexing of smFISH (i.e., the number of different RNA species that can be simultaneously quantified within the same cell) have been developed (8-13). Most of these approaches take advantage of color multiplexing, which has allowed a few tens...

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Section: Significancementioning

confidence: 99%

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Moffitt

Hao

Bambah-Mukku

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

233

249

View full text Add to dashboard Cite

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“…The physical properties of the lipid bilayer prevents large macromolecules for example, antibodies, from readily diffusing deep into the tissue whilst the molecular heterogeneity of the lipids themselves alter the refractive index of the tissue, further perturbing the excitation and emission of light from fluorescently-labelled probes (Richardson & Lichtman 2015). To overcome these problems, "clearing" methods such as CLARITY, PARS, CUBIC, SeeDB, ScalA2, iDISCO and 3DISCO have been developed (Susaki et al 2015;Bin Yang et al 2014;Renier et al 2014;Tomer et al 2014;Susaki et al 2014;Ke et al 2013;Becker et al 2012;Hama et al 2011). …”

Section: Novel Deep Tissue Imaging Methodsmentioning

confidence: 99%

Novel imaging tools for investigating the role of immune signalling in the brain

Jacobsen

Parker

Everest‐Dass

et al. 2016

Brain, Behavior, and Immunity

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In all cases accepted manuscripts should:  link to the formal publication via its DOI  bear a CC-BY-NC-ND license -this is easy to do, click here to find out how  if aggregated with other manuscripts, for example in a repository or other site, be shared in alignment with our hosting policy  not be added to or enhanced in any way to appear more like, or to substitute for, the published journal article AbstractThe importance of neuro-immune interactions in both physiological and pathophysiological states cannot be understated. As our appreciation for the neuroimmune nature of the brain and spinal cord grows, so does our need to extend the spatial and temporal resolution of our molecular analysis techniques. Current imaging technologies applied to investigate the actions of the neuroimmune system in both health and disease states have been adapted from the fields of immunology and neuroscience and are inherently limited by their semiquantitative nature, loss of spatial resolution, photobleaching and detection sensitivity. Thus, the development of innovative methods which overcome these limitations are crucial for imaging and quantifying acute and chronic neuroimmune responses. Therefore, this review aims to convey novel and complementary imaging technologies in a form accessible to medical scientists engaging in neuroimmune research.

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“…VIS cameras are used to estimate the true color of each pixel because of presence three color sensors (red, blue and green) in camera array. Analysis of color parameters, geometric and morphological characteristics can be done by the operation of VIS cameras in the phenotyping system (Tessmer et al, 2013, Yang et al, 2014. Near-infrared (NIR) light can be recognized by infrared (IR) cameras with usage for night imaging processing (Matos et al, 2014).…”

Section: Integration Of Imaging Techniques In High Throughput Phenotymentioning

confidence: 99%

Applying hyperspectral imaging to explore natural plant diversity towards improving salt stress tolerance

Sytar

Brestič

Živčák

et al. 2017

Science of The Total Environment

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• Salinity represents an abiotic stress constraint affecting growth and productivity of plants • Better solutions is to improve the level of salt resistance using natural genetic variability within crop species • Phenomic methodology employing different non-invasive imaging systems for detecting quantitative and qualitative changes caused by salt stress at the whole plant and canopy level. Hyperspectral imaging techniques provide unique opportunities for fast and reliable evaluation of numerous characteristics associated both with various structural, biochemical and physiological traits • Salt-soil-plant interaction and sustainable coastal agriculture need powerful phenotyping tools Salinity represents an abiotic stress constraint affecting growth and productivity of plants in many regions of the world. One of the possible solutions is to improve the level of salt resistance using natural genetic variability within crop species. In the context of recent knowledge on salt stress effects and mechanisms of salt tolerance, this review present useful phenomic approach employing different non-invasive imaging systems for detection of quantitative and qualitative changes caused by salt stress at the plant and canopy level. The focus is put on hyperspectral imaging technique, which provides unique opportunities for fast and reliable estimate of numerous characteristics associated both with various structural, biochemical Science of the Total Environment 578 (2017) 90-99 ⁎ Corresponding authors at:

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Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

Cited by 808 publications

References 38 publications

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Novel imaging tools for investigating the role of immune signalling in the brain

Applying hyperspectral imaging to explore natural plant diversity towards improving salt stress tolerance

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