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Posted: Jan 30, 2015
The amazing disappearing mouse
(Nanowerk News) In a Petri dish culture, cells and tissue are sufficiently transparent that they can be readily explored using any of a variety of microscopy techniques. Whole organs, however, are opaque under light microscopy, and it has proved challenging to find a way to clarify tissue to the extent needed to permit microscope observations of individual cells deep inside an animal. A research team led by Hiroki Ueda and Kazuki Tainaka from the Laboratory for Synthetic Biology at the RIKEN Quantitative Biology Center has now developed a remarkably effective tissue-clearing technique that promises to allow direct microscopy studies of deep tissue in organs and even whole animals (Cell, "Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization").
The research team previously discovered a mixture of certain aminoalcohols that causes brain tissue to become clear and glassy, breaking down the dense lipids that block and scatter light. They combined this chemical treatment with a set of image-processing tools, and the resulting ‘clear, unobstructed brain imaging cocktails and computational analysis’ (CUBIC) method enabled easy visualization of fluorescently labeled proteins and cells deep within the brain.
A number of approaches have been developed to break down lipids in fixed tissues, but most of these techniques are optimized for the lipid-rich brain. Although a promising clearing strategy that works at the whole-body scale has also been reported, its utility is limited by its inability to remove naturally occurring pigments like heme, which gives red blood cells their distinctive color. “As pigments are a major source of light absorption,” says Tainaka, “whole organs treated by this method still appeared opaque.”
A moment of clarity
As Ueda’s team began to experiment with the CUBIC treatment of different organs, they made the unexpected discovery that these organs began to lose their color over the course of treatment. At the same time, the aminoalcohol mixture bathing these samples changed in color from clear to green, suggesting the presence of iron-laden heme. “This observation led us to hypothesize that the CUBIC cocktail could solubilize and eliminate endogenous heme from blood-infused tissues,” says Tainaka.
Working with colleague Shimpei Kubota from the University of Tokyo, Tainaka subsequently determined that this ‘transparentization’ treatment was broadly applicable to a wide variety of organs. The pair examined nearly a dozen different whole organs, including the heart, liver and lungs, and found that all of these could be effectively clarified and decolorized after ten days of treatment with the CUBIC cocktail (Fig. 1). Remarkably, with a slightly longer treatment time, an entire infant mouse could be rendered largely transparent—its skeleton could be observed beneath the clarified organs and muscle. The researchers also achieved the same effect in adult mice, although these were too large to visualize under a microscope in their entirety.
The CUBIC mixture works in part by maintaining a moderately alkaline environment that promotes the release of heme. This alkaline environment also supports labeling, such as with green fluorescent protein, which allows biologists to introduce genetically encoded visible ‘tags’ for specific proteins, cells or tissues of interest. Using green fluorescent protein and other fluorescent labels, Tainaka, Kubota and their colleagues were able to directly visualize the fine structure of the chambers of the heart, the bronchial passageways of the lungs, and the vascular structures of the liver. In all of these cases, computational processing of the resulting data made it possible to clearly distinguish individual fluorescently labeled cells within the three-dimensional environment of a given organ.
A clear difference
This new clearing method could help scientists observe disease-related disruptions within the body in far greater detail than is possible using isolated tissue slices. To demonstrate this potential, Tainaka and Kubota used CUBIC to assess pancreatic pathology in a mouse model of type I diabetes. Patients with this disease produce antibodies that destroy the insulin-producing beta cells within a pancreatic structure called the Langerhans islets, and thus lose the capacity to regulate their blood sugar. A similar state can be induced by treating animals with a chemical called streptozotocin (STZ), which also selectively kills beta cells. CUBIC analysis confirmed that STZ treatment diminished both islet volume and cell count, and that mice with larger islets were more vulnerable to STZ-induced diabetes (Fig. 2).
Figure 2: Computational analysis of the clarified and decolorized mouse pancreas (top left) makes it possible to identify the insulin-producing Langerhans islets (top right). These can then be mapped three-dimensionally (bottom left) either alone or in the context of the entire organ (bottom right). (click on image to enlarge)
Although these findings are not surprising, they demonstrate the capacity of this imaging technique to peer inside mysteries associated with other disease states. “Since our technique enables the imaging of an entire body without sectioning,” says Tainaka, “it would be suitable for exploring unknown cellular aberrations induced by cancer metastasis and immunological response, or for revealing unknown cellular interactions between organs.”
Over the past few decades, scientists have generated transgenic animals that selectively produce fluorescently labeled proteins in a broad range of cell types. However, CUBIC is not limited to these models, and the researchers demonstrated that similar results could also be obtained with non-genetically modified mice by treating clarified organs with dye-labeled antibodies that recognize a specific cellular protein of interest. Using this approach, also known as immunohistochemistry, virtually any disease state that can be examined by conventional pathology techniques should also be accessible using CUBIC.
By drawing on powerful computational tools for three-dimensional image reconstruction and single-cell tracing, Tainaka and his colleagues hope to use CUBIC to more thoroughly trace the development of diseases that progress over extended periods of time, such as following the stages of malignancy in a growing tumor or the gradual accumulation of damage in different tissues arising from autoimmune disorders. “We could apply this technique to achieve a systems-level understanding of cellular disease mechanisms,” says Tainaka.