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Jul 31, 2014

The Body Transparent: Tissue Clearing Lays Fine Structures Bare

The Body Transparent: Tissue Clearing Lays Fine Structures Bare

A 3D visualization of intact intestine tissue, obtained through whole-body clearing and staining. [Bin Yang and Viviana Gradinaru]

  • Walt Whitman’s Body Electric has nothing on whole-body clearing, which might be called the Body Transparent. Whereas Whitman conjoined the “parts and poems of the body,” even the “thin red jellies within,” to flesh out a vision of connectedness, whole-body clearing—a kind of lipid-leaching process—enables the mapping and phenotyping of normal and pathological elements within intact organs and bodies. In doing so, whole-body clearing realizes one of the learn’d biologist’s dearest dreams—the visualization of long-range connections between cells as well as fine-grained cellular structures.

    Processes capable of rendering tissues optically transparent have been around since the 1800s, but they tend to damage tissue, distorting the body’s finer structures. Worse, these processes frequently rely on chemistries that are incompatible with labeling and staining methodologies, and so leave many structures of interest invisible.

    These problems have been largely overcome by newer processes, such as CLARITY, which stands for Clear, Lipid-exchanged, Acrylamide-hybridized Rigid, Imaging/immunostaining compatible, Tissue hYdrogel. CLARITY, which was developed at the Deisseroth Lab at Stanford, and similar approaches provide hydrogel embedding to stabilize tissue structures, fluorescent protein-compatible clearing reagents, and imaging procedures for large tissue samples. While such approaches have been used to generate 3D images of complex, long-distance cellular interactions, they have been limited to the brain or embryos.

    Taking approaches such as CLARITY a step further, a California Institute of Technology research team led by Viviana Gradinaru, Ph.D., has developed methods for making opaque organs, bodies, and human tissue biopsies transparent, while keeping the cellular structures and connections intact. These methods include PACT (PAssive Clarity Technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (Refractive Index Matching Solution), a refractive index matching media for imaging thick tissue; and PARS (Perfusion-Assisted agent Release in Situ), a method for whole-body clearing and immunolabeling.

    The details appeared July 31 in the journal Cell, in an article entitled, “Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing.” According to Dr. Gradinaru, PACT, RIMS, and PARS together represent the first attempt to perform whole-body clearing, as opposed to first extracting and then clearing organs outside the adult body.

    “We here propose a methodology to facilitate fast, whole-brain and whole-body clearing using the circulatory system or the cerebrospinal fluid route to directly deliver clarifying agents,” wrote the Cell article’s authors. “We demonstrate … that opaque, intact, whole-organisms [can be transformed] into optically transparent, fluorescently labeled samples for visualization with conventional confocal microscopy and phenotypic analysis at the cellular, subcellular, and even single-molecule transcripts level.”

    “Our methodology has the potential to accelerate any scientific endeavor that would benefit from whole-organism mapping, including the study of how peripheral nerves and organs can profoundly affect cognition and mental processing, and vice versa,” asserted Dr. Gradinaru. “Our easy-to-use tissue clearing protocols, which employ readily available and cost-effective reagents and equipment, will make the subcellular interrogation of large tissue samples an accessible undertaking within the broader research and clinical communities.”


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