Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
There has, perhaps, never been a better time to be a neuroscientist. An influx of money, computational muscle and technology advances have combined to expose the brain to greater scrutiny than ever before. The researchers compiling the Allen Brain Atlas, for instance, are using viruses that encode fluorescent proteins to trace neural projections throughout the mouse brain at the microscopic level, while other neuroanatomists use serial electron microscopy approaches to map connections at the nanoscale.
The data from these projects are rich and publicly available, and they are beautiful to behold. But all of them represent a trade-off of sorts. It simply isn’t possible to image the cellular anatomy of the brain at once—the tissue is too thick, and its rich lipid content scatters light. So these projects require slicing brain tissue into thin sections, each of which is then imaged and reassembled to build a 3D representation of the data. Yet the simple act of slicing and reconstructing tissue can lead to distortion and artifacts among the neuronal arbors' fine projections.
Recently, researchers have devised strategies to make brains optically transparent, thereby obviating the need for thin sectioning. One, published in 2013 by Karl Deisseroth’s lab at Stanford University, is called CLARITY [1,2]. The method stabilizes tissue in an acrylamide scaffold and then removes lipids by passive or electrophoretic diffusion. The approach has two advantages: First, the tissue structure and macromolecular localization are maintained, producing essentially a 3D molecular echo of the original tissue. Second, "clarified" tissue can be repeatedly stained for molecular markers, something that isn’t generally possible with other clearing methods.
Here, Biocompare takes a look at how CLARITY works and the tools you’ll need to use it.
CLARITY protocol
According to Viviana Gradinaru, assistant professor of biology and biological engineering at the California Institute of Technology, who helped develop the method while in Deisseroth’s lab, CLARITY was conceived to extend to mouse studies the benefits of C. elegans and zebrafish: optical transparency. “The difficulty is the lipids,” Gradinaru explains—lipids in the brain make the tissue opaque and light-scattering. But lipids also provide much-needed structural support, she says, “like a skeleton of a building. If you remove them, your tissue flattens out, [and] you don’t maintain shape.”
The trick, therefore, was to devise a way to remove the lipids while maintaining the structure. The solution: to effectively embed the tissue in a polyacrylamide gel.
CLARITY has three basic steps. First, the tissue is infused with hydrogel monomers—originally acrylamide and bisacrylamide, the ingredients of protein gel electrophoresis—in the presence of formaldehyde, which crosslinks proteins and nucleic acids. After one to three days, the temperature is increased to polymerize the acrylamide, thereby effectively scaffolding the brain tissue.
The second step is lipid removal, originally via a process called “electrophoretic tissue clearing,” in which SDS micelles are driven through the tissue in an electric field over the course of several days. Finally, the resulting tissue is stained using fluorescent antibodies or nucleic acid probes (a process that can take up to two weeks) and imaged.
The process sounds straightforward, but some researchers still struggle with the process, as evidenced by traffic at forum.claritytechniques.org. So researchers have developed several modifications.
Ilya Bezprozvanny, professor of physiology at the University of Texas Southwestern Medical Center in Dallas, for instance, found that the electrophoretic clearing step was particularly difficult to get right. “You need a specialized chamber, and the conditions have to be just right, and it takes a long time,” Bezprozvanny explains. So he developed a simple alternative, called CLARITY2, in which the brain is sectioned into 1- to 1.5-mm sections and cleared via passive diffusion in 50-ml conical tubes [3].
If that seems like a regression to the tissue slicing CLARITY was designed to replace, it is, and it isn’t. Traditional thin sections are typically 20 to 30 microns thick, whereas these sections measure a millimeter or more. “Our method is sort of in between,” Bezprozvanny says. “You don’t have the whole brain, but thick slices. And it’s very likely that the structure [researchers are interested in] will be contained in one or maybe two slices.”
Gradinaru has developed a more substantial technical modification, called PACT (passive CLARITY technique) [4]. As in CLARITY2, PACT eliminates the electrophoretic clearing step, as the process can heat tissue and cause it to brown. Instead, the method uses passive clearing—a change made possible by, among other things, the removal of bisacrylamide, thereby producing a less-crosslinked hydrogel, and higher SDS concentrations. Gradinaru also extended the technique to stabilize and clarify not just isolated brains but entire rodents using pumped perfusion through the circulatory system—a method called PARS (perfusion-assisted agent release in situ).
In total, the new process takes one to two weeks, Gradinaru says—about the same as the original CLARITY protocol. But in that time, the entire organism can be clarified, meaning researchers can visualize any organ they wish; indeed, in her paper Gradinaru imaged and stained multiple tissues, including rodent heart, kidney, lung and spinal cord.
“The speed wasn’t improved that much; what was improved was the quality of tissue. Because if you leave tissue in an electrical field for two weeks, the chance of damage is quite high,” she says.
Microscopy considerations
CLARITY is compatible with confocal, multiphoton and light-sheet microscopy, among others. But given the thickness of the specimens and the nature of the reagents used, the technique does require some specialized microscopy tools. Long working distance objectives (8 mm or more, Gradinaru says) are a must, but neither standard water- nor oil-immersion optics will suffice, as both can produce distortions because of the difference in refractive index with the clarified sample.
“If you don’t match the refractive index, you’ll get a suboptimal picture,” says Bernd Sägmüller, director of confocal laser scanning microscopy at Leica Microsystems. “You need alignment between the optical properties of the sample, the immersion medium and the objective, otherwise, you get spherical aberration and color distortion.”
In the original CLARITY paper, the authors immersed the sample in a solution called FocusClear, which matched the refractive index of the sample itself, and then imaged with a water-immersion objective. But FocusClear is expensive and not readily available, Gradinaru notes, so her more recent paper describes a DIY alternative called RIMS (refractive index matching solution), which “provided a >10-fold reduction in mounting costs” relative to FocusClear, according to the publication.
Several microscopy vendors, including Leica, now offer dedicated objectives to accommodate CLARITY (and related imaging techniques), as described in a recent Nature Methods review [5]. Leica's, for instance, is the HC FLUOTAR L 25x/1.00 IMM motCORR™ VISIR. “When the [CLARITY] process is done, your tissue is stored in a special immersion medium,” Sägmüller explains. “Our objective matches exactly the optical properties of that medium.”
Other than that, the technique requires little in the way of specialized tools.
If you wish to try the technique, Gradinaru offers these tips. First, “Do not overclear. That’s important. Because transparency to the eye is not necessarily the goal.” She recommends checking the sample under the microscope throughout the clearing process to ensure endogenous fluorescence, if applicable, is still visible.
Also, keep your samples at room temperature, Gradinaru says. Users may be inclined to store their samples at 4oC either during preparation or following imaging. But at that temperature, she notes, CLARITY solutions precipitate, causing turbidity in the tissue.
And finally, be prepared to do some number crunching. “There’s going to be a lot of imaging and data,” she says. “That can turn into a bottleneck, if it’s not properly planned for.”
References
[1] Chung, K, et al., “Structural and molecular interrogation of intact biological systems,” Nature, 497:332-7, 2013. [PubMed: 23575631]
[2] Tomer, R, et al., “Advanced CLARITY for rapid and high-resolution imaging of intact tissues,” Nat Protocols, 9:1682–97, 2014. [PubMed: 24945384]
[3] Poguzhelskaya, E, et al., “Simplified method to perform CLARITY imaging,” Mol Neurodegeneration, 9:19, 2014. [PubMed: 24885504]
[4] Yang, B, et al., “Single-cell phenotyping within transparent intact tissue through whole-body clearing,” Cell, doi:10.1016/j.cell.2014.07.017, 2014. [PubMed: 25088144]
[5] Marx, V, “Microscopy: Seeing through tissue,” Nat Methods, 11:1209-14, 2014. [PubMed: 25423017]
Image: Caption: CLARITY allows molecular analysis of the intact brain. Each color represents a different molecular label; this labeling can happen after the brain is clarified but still fully intact. Hippocampus is shown, a structure important for many important roles including learning, memory, and emotion. Credit: Kwanghun Chung and Karl Deisseroth, Howard Hughes Medical Institute/Stanford University (Nature, 497:332, 2013)