Several years ago, a colleague came to Japanese researcher Atsushi Miyawaki with a simple observation. While performing a Western blot experiment with gels, he noticed that the light scattering properties of the gels disappeared when submerged in a solution containing the common laboratory compound urea. So, Miyawaki wondered if a urea solution could also reduce the light scattering of tissues, which would allow biologists to peer deeper into an organism to observe its hidden physiology. And it did.
While Miyawaki’s tissue-clearing urea reagent will allow biologists to peer further into the depths of tissue of organisms, nuerobiologists are particularly interested because the reagent promises to reveal the structure of the nervous system without the complications or limitations of traditional sectioning techniques. But because this reagent provides clarity at unprecedented tissue depth, a new rate-limiting step is the microscope objectives, bringing microscopy companies a new challenge.
Problems Seeing Clearly
At his Harvard lab, Lichtman and his team want to understand the connections between competing neurons. In the organisms that they study, these cells are more often than not grouped together in 3-D masses within the brain, spinal cord, muscle, or the peripheral nervous system. So, in order to understand these connections, Lichtman’s lab uses approaches to image not just single planes of the organism but the 3-D structures hidden beneath the surface.
One of these 3-D imaging approaches is serial sectioning, which was introduced in 1980 by Peter Sterling and colleagues at the University of Pennsylvania (1). In this technique, an object is sliced into many thin sections, and those sections are individually imaged using electron microscopy. The images are then combined to reconstruct the neural tissue. “But it’s extremely tedious, and there are many, many technical problems with doing serial sections,” says Lichtman.
In contrast, the alternative—optical sectioning—does not require cutting the tissue. Instead, these microscopy techniques provide information about a particular optical plane within a tissue sample by eliminating the information from the optical planes above and below. Widely used optical sectioning methods include confocal microscopy and two-photon excitation microscopy, both of which rely on fluorescent labeling technology to focus on imaging one optical plane at a time. Afterward, these images are then reconstructed to provide the 3-D structure without damaging the tissue.
But the problem with optical sectioning is that as researchers image deeper and deeper into the tissue, the material above the targeted plane disrupts the signal. One reason is light scattering. “If you shine light through a material that is milky, that lets light go through but scatters it, it doesn’t come out looking like it came in,” says Lichtman. “You put a laser on one side, and at the other side, you just get a glow as the laser beam is bounced all over the place. The exact same thing happens when you image deep into the brain.”
As a result, a good deal of interest and effort has been put forth to develop ways to clear tissue. One way to do clear tissue is to remove all the water from the sample, but without water, the fluorescent proteins lose their fluorescence. Although life science suppliers market several clearing agents that are designed to maintain fluorescence proteins, none have been a perfect solution.
“Most of them, we found, were not very good. They either ruined the fluorescence of the fluorescence protein or they didn’t clear well,” says Lichtman. In the end, an agent that maintains the fluorescence and clears the tissue remained elusive.
“Mechanical sectioning is laborious and very hard to make large-scale 3-D reconstructions of brain tissue,” says Miyawaki. “But with optical sectioning approach, tissue penetration has always been a challenging problem because deep imaging is limited by light scattering. We were very eager to solve this problem.”
So, when Miyawaki’s colleague came to him with the observation that a urea solution could diminish the light-scattering properties of gels, he was very interested. Urea is a common and inexpensive laboratory reagent used to denature proteins. In animals, urea is a breakdown metabolite of nitrogenous compounds, carrying waste nitrogen out of the body through urine.
After verifying the tissue-clearing properties of the urea solution, Miyawaki and colleagues spent years developing a formula that would achieve excellent clarity while preserving the function of fluorescent proteins. In a paper published in Nature Neuroscience in September 2011, using their tissue-clearing reagent named Scale, Miyawaki’s team imaged fluorescently labeled neurons in a fixed mouse brain at a depth of 4mm (2).
“It aims to reduce the scattering by a considerable amount, and importantly, and this is the thing that makes it better than most clearing reagents, it does so in a way that does not disrupt the ability of the fluorescence proteins to fluoresce,” says Lichtman.
But even after years of development, the technique is still difficult to get to work perfectly. For one thing, it’s much slower than some other clearing reagents such as a mixture of benzyl-alcohol and benzyl-benzoate (BABB) that can clear tissue in hours. Tissue samples must soak in the urea solution for a long time—sometimes months—to achieve the required clearing. The time depends on the type of tissue as well as the targeted depth and clarity.
“We are very patient people,” says Miyawaki. “We left the samples alone for a couple of weeks to find the optical clearing effect of the urea solution. So, we are not hasty at all; we almost forgot what we were doing.”
Because the reagent is so easy and inexpensive to make, several labs are already trying it. Back at Harvard, Lichtman is attempting to use the Scale reagent in his Brainbow technique, which uses multiple fluorescent proteins to differentiate neurons in brain tissue samples. "Part of the limitations of the Brainbow technique at the moment is scattering, so having tools to clear the brain will be really valuable,” says Litchman. “We’re trying very hard to make this work well in our tissues.”
In contrast to commercially available clearing reagents, the Scale solution is not proprietary, something that Miyawaki feels particularly proud of. Because the ingredients of the solution are presented in the paper, anybody can modify the formula as needed for their specific samples. By being open about the formula, Miyawaki hopes to give researchers the flexibility to develop new applications and further evolve microscopy.
Furthermore, because of RIKEN’s and Miyawaki’s long working relationship with Japanese microscopy manufacturer Olympus Corporation, the Scale reagent is now commercially available for laboratories that do not have the time or the means to prepare the solution themselves. The Olympus Scaleview-A2 clearing reagent allows a greater number of researchers access to the technique.
As a result of the wide availability of the Scale reagent, the rate-limiting step in deep-tissue imaging has now fundamentally shifted. “If I can get my tissue sample clear to 1 cm, it wouldn’t be that useful to me,” says Lichtman. “I don’t have a microscope objective that could focus in that far.”
Traditional confocal and two-photon microscopy objectives have working distances in the range of hundreds of microns. If a researcher tried to focus any deeper into the tissue than that, the objective would bump into the tissue. So, even if the Scale reagent could clear tissue beyond the hundreds of micron scale, it wouldn’t benefit most researchers with objectives with improved working distance.
So, Miyawaki asked Olympus to develop and optimize a refractive-index long-distance working objective to complement his reagent. Furthermore, Miyawaki wanted these objectives to be widely available to researchers, so Olympus commercialized them and began distributing them across the globe. Designed specifically to be used with the Scaleview-A2 reagent and the Olympus FluoView FV1000-MPE multiphoton microscope, the two new 25x long-working-distance objectives can image structures 4-mm and 8-mm deep, respectively.
“It’s not just having a long-distance objective, but what you need is something that can make use of the deepest penetration possible, which is multiphoton microscopy and maintaining high resolution and high numerical aperture,” says confocal sales representative Brendan Brinkman from Olympus.
These two objectives provide that high resolution and high numerical aperture, which will allow connectomics projects to trace filaments throughout the brain in depth. With the development of the Scale reagent and long-distance objectives, the possibility of doing complete connectivity maps with light microscopy not requiring sectioning becomes clearer.
In addition, Miyawaki’s lab is currently developing other mild clearing reagents, hoping to adapt the Scale technique to live tissue, organs, and animals. “We do not need 100% transparency, 50% is good enough, but we need to preserve the natural activity and viability,” says Miyawaki. “So for that, a mild solution is very critical.” While the current version of the Scale reagent contains urea, a metabolic waste product, an agent for live tissue would require other candidates, but so far, Miyawaki is not happy with any results.
“Until last September, a 2-mm objective was the longest that you could buy, but now you can purchase a 4-mm and, using that lens, look at the structure down to exactly 4 mm below the brain surface,” says Miyawaki. “This technique is very powerful, and I think this technique and surrounding techniques should evolve quickly from now on. That is my hope.”
- Stevens JK, Davis TL, Friedman N, Sterling P. 1980. A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections. Brain Res Rev 2:265–293.
- Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. 2011. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci. 14:1481-8.