Flushing out the fatty fog

PASADENA, Calif.—According to legend, Renoir’s last words about painting were, “I think I’m beginning to see…” and life sciences may be saying the same soon. In general, our knowledge of complex systems such as those found in biology is limited by our inability to actually “see” things in those systems. Researchers who study developmental problems and disease, in particular, are often limited by their inability to look inside an organism to figure out exactly what went wrong, and when.

“Large volumes of tissue are not optically transparent,” notes Viviana Gradinaru, an assistant professor of biology at the California Institute of Technology (Caltech) and the principal investigator whose team has developed new techniques for doing just that, which are explained in a paper appearing in the journal Cell. Lipids throughout cells provide structural support, but they also prevent light from passing through the cells. “So, if we need to see individual cells within a large volume of tissue”—within a mouse kidney, for example, or a human tumor biopsy—“we have to slice the tissue very thin, separately image each slice with a microscope and put all of the images back together with a computer. It’s a very time-consuming process, and it is error-prone, especially if you look to map long axons or sparse cell populations such as stem cells or tumor cells,” she says.

But that may be about to change dramatically.

The researchers came up with a way to circumvent this long process by making an organism’s entire body transparent, so that it can be peered through—in 3D—using standard optical methods such as confocal microscopy.

The new approach builds off a technique known as CLARITY that was previously developed by Gradinaru and her collaborators to create a transparent whole-brain specimen. With the CLARITY method, a rodent brain is infused with a solution of lipid-dissolving detergents and hydrogel—a water-based polymer that provides structural support—thus “clearing” the tissue but leaving its three-dimensional architecture intact for study.

The refined technique optimizes the CLARITY concept so that it can be used to clear other organs besides the brain, and even whole organisms. By making use of an organism’s own network of blood vessels, Gradinaru and her colleagues—including co-authors Bin Yang and postdoctoral scholar Jennifer Treweek—can quickly deliver the lipid-dissolving hydrogel and chemical solution throughout the body in a process they call PARS or perfusion-assisted agent release in situ.

The abstract published in Cell notes that, “Understanding the structure-function relationships at cellular, circuit and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging.” The paper presents “techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.”

Once an organ or whole body has been made transparent, standard microscopy techniques can be used to view single cells that are genetically marked with fluorescent proteins. Even without such genetically introduced fluorescent proteins, however, the PARS technique can be used to deliver stains and dyes to individual cell types of interest. When whole-body clearing is not necessary, the method works just as well on individual organs by using the PACT technique.

To find out if stripping the lipids from cells also removes other potential molecules of interest—such as proteins, DNA and RNA—Gradinaru and her team collaborated with Long Cai, an assistant professor of chemistry at Caltech, and his lab. The two groups found that strands of RNA are indeed still present and can be detected with single-molecule resolution in the cells of the transparent organisms.

The Cell paper focuses on the use of PACT and PARS as research tools for studying disease and development in research organisms. However, Gradinaru and her University of California, Los Angeles, collaborator Rajan Kulkarni have already found a diagnostic medical application for the methods. Using the techniques on a biopsy from a human skin tumor, the researchers were able to view the distribution of individual tumor cells within a tissue mass. In the future, Gradinaru says, the methods could be used in the clinic for the rapid detection of cancer cells in biopsy samples.

The ability to make an entire organism transparent while retaining its structural and genetic integrity has broad-ranging applications, Gradinaru says. For example, the neurons of the peripheral nervous system could be mapped throughout a whole body, as could the distribution of viruses, such as HIV, in an animal model.

Gradinaru also leads Caltech’s Beckman Institute BIONIC center for optogenetics and tissue clearing and plans to offer training sessions to researchers interested in learning how to use PACT and PARS in their own labs.

“I think these new techniques are very practical for many fields in biology,” she says. “When you can just look through an organism for the exact cells or fine axons you want to see—without slicing and realigning individual sections—it frees up the time of the researcher. That means there is more time to answer the big questions.”