Clarifying Tissue Clearing

Richardson, Douglas S.; Lichtman, Jeff W.

doi:10.1016/j.cell.2015.06.067

Cited by 952 publications

(1,059 citation statements)

References 63 publications

(95 reference statements)

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“…It is also likely that other clearing solutions could be substituted for BABB (Richardson & Lichtmann, 2015), and that clearing of live larvae may be possible using the refractive‐index tunable and nontoxic clearing method recently by Boothe et al . (Boothe et al ., 2017).…”

Section: Discussionmentioning

confidence: 99%

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

McConnell

Amos

2018

Journal of Microscopy

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SummaryIn a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently‐stained 12.5‐day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.

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Section: Discussionmentioning

confidence: 99%

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

McConnell

Amos

2018

Journal of Microscopy

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“…Many of the limitations, such as difficulty binding to and visualizing antigens, stem from the lipid membrane. 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%

“…This enables light to penetrate deeper, facilitating the imaging of whole organs (Richardson & Lichtman 2015).…”

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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“…50 We find that lScaleSQ induces sample swelling, likely due to the hyperhydration caused by the urea. 47 All three processes significantly increase the average transmitted light intensity through the sample, with lSeeDB and lScaleSQ offering a greater increase in the transmitted intensity than lClear T2 .…”

Section: A Clearing Increases Fluorescence Imaging Penetration Depthmentioning

confidence: 99%

“…35 This shrinkage-expansion process for lClear T2 and lScaleSQ(0) is likely due to an initial osmotic shrinkage followed by swelling due to hyperhydration; formamide (which is similar to urea) may clear tissues by a similar hyperhydration mechanism. 47 Awareness of responses like this could be important to the users of this clearing method as the shrinkage-expansion process has the potential to induce imaging artefacts. Our microfluidic implementation facilitates the monitoring required to discover these types of effects.…”

Section: On-chip Clearing Is Rapid and Permits Monitoring Of 3-d Cmentioning

confidence: 99%

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

et al. 2016

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Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 lm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear T2 , and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear T2 ; however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This onchip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. Published by AIP Publishing. [http://dx

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Clarifying Tissue Clearing

Cited by 952 publications

References 63 publications

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

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

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

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