Single-cell phenotyping within transparent intact tissue

Developers

Bin Yang, Jennifer B. Treweek, Long Cai, Viviana Gradinaru, etc.

Description of the technology

The proposed technology enables to visualize the cellular architecture of entire organs using techniques for making tissues transparent, allowing to see organs and inner structures of animals at depths of millimeters to centimeters by using microscopes.

For «optical clearing» of animal tissues, the special technique is applied, named CLARITY, which in original version involves the stabilization of specimens with an acrylamide hydrogel and then the depletion of them with lipids. In the frameworks of this technology, the modified version of CLARITY is used. In process of this modified version, hydrogel monomers and lipid-clearing agents are pumped into animals directly through the vasculature and the cerebrospinal fluid. The advantage of this approach is speed, as large organs and even entire mice can be cleared within a few days, thereby exposing their cellular structure with intact connectivity.

The technology includes PACT, a protocol for passive tissue clearing and immunostaining of intact organs; RIMS, a refractive index matching media for imaging thick tissue; and PARS, a method for whole-body clearing and immunolabeling.

The demonstration of applicability of the technology was carried out by the clearing of a variety of mouse organs, including brain, heart, lung, intestine and kidney as well as the body of a whole mouse. It was shown thereby that PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. It was also demonstrated that PACT, RIMS, and PARS are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.

Practical application

The technology allows the observing of cells and molecules within the intact architecture of the tissues and organs, which is crucial for understanding of the structure and function of complex biological systems. It is applicable to identify the structure-function relationships at cellular, circuit, and organ-wide scale and construct 3D anatomical and phenotypical maps.

Besides, the technology enables systematic identification of cell types throughout the volume monitored during previous live imaging. This identification is carried out by staining of key transcription factors and other genes of interest or by high-resolution imaging of cell morphologies. With this information, one could systematically investigate the behavior and interactions of different cell populations, for example, how they build individual tissues and organs during development, or reveal the repertoire of neurotransmitters, receptors and channels in neural tissues following high-speed calcium imaging.

Laboratories

• Division of Biology and Biological Engineering, California Institute of Technology, Pasadena (USA)
• Division of Dermatology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles (USA)
• Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena (USA)

Links

Publications

• Höckendorf, B., Lavis, L.D., Keller, P.J. «Making biology transparent» 32.11 Nature Biotechnology, (2014): 1104–1105.
• Yang, B. et al. «Single-cell phenotyping within transparent intact tissue through whole-body clearing." 158.4 Cell. (2014): 945−958.
• Chung, K. et al. «Structural and molecular interrogation of intact biological systems." 497 Nature, (2013): 332−337.
• Gradinaru, v. et al. «Molecular and cellular approaches for diversifying and extending optogenetics." 141.1 Cell. (2010): 154−165.