An important challenge in microscopy is the development of high-resolution light microscopy methods to label and image cell populations in three dimensions. The ability to achieve this deep into intact specimens is limited by light scattering. Modern technologies, such as two-photon excitation fluorescence microscopy, allow examination of structures at distances of hundreds of micrometers below the surface but are insufficient to image and reconstruct large cell populations that are millimeters in scale and deeper below the surface. Whereas light scattering can be reduced by optical clearing, most of these reagents exhibit limitations such as the quenching of fluorescence. Recently, a clearing agent that spectacularly alleviates these major limitations was developed by Hiroshi Hama, Hiroshi Kurokawa, Hiroyuki Kawano, Ryoko Ando, Tomomi Shimogori, Hisayori Noda, Kiyoko Fukami, Asako Sakaue-Sawano, and Atsushi Miyawaki [Reference Hama, Kurokawa, Kawano, Ando, Shimogori, Noda, Fukami, Sakaue-Sawano and Migawaki1]. They developed a clearing reagent called Scale that renders mouse brains and embryos transparent while completely preserving fluorescent signals from labeled cells!

The discovery of the Scale reagent started with the serendipitous observation that polyvinylidene fluoride membranes became transparent when soaked in 4M urea, a reagent that promotes hydration of biological samples. This result inspired Hama et al. to search for an optimal reagent to clear fixed biological samples for light microscopy. They experimented with various concentrations of urea and other ingredients. The most effective solution was composed of 4M urea, 10% glycol, and 0.1% Triton X-100 (a detergent). They quantitatively compared transparency through fixed brain slices using this formulation of Scale and other available clearing agents. Specimens treated with Scale permitted substantially more light through in the visible and infrared ranges. Intact fixed mouse brains (and whole mouse embryos) became relatively transparent after incubating in Scale for 2 weeks or more. Specimens were seen to expand (about 1.25-fold linearly), but careful experiments showed that tissue expansion was isotropic and homogeneous. Interestingly, the term Scale was chosen as the code word for the project because the word sounds like “something transparent” in Japanese.

Hama et al. next investigated if the capability for fluorescence imaging is retained in Scale-treated specimens because this is critical for imaging individual neurons in the brain. Experiments in vitro and with cultured cells showed that enhanced green fluorescent protein signal was not substantially decreased by treatment with Scale, whereas a conventional clearing agent dimmed the signal over time. They next treated whole brains of transgenic mice with yellow fluorescent protein. Certain subpopulations of neurons in these brains are known to fluoresce. The whole fixed brain showed homogeneous fluorescence; but, when one half was treated with Scale and the other half treated with a conventional clearing agent, fluorescence was only retained in the former.

Using one- and two-photon excitation fluorescence microscopy, Hama et al. demonstrated that three-dimensional neuronal structures could be reconstructed using sophisticated imaging software. They found that in brains cleared with Scale, the imaging depth was limited by the working distance of the objective lens. They even asked the lens manufacturer Olympus to develop a customized 25× objective lens with a working distance of 4 mm and a numerical aperture (1.0) that allows for high resolution.

Using various formulations of Scale, Hama et al. could vary the time required for clearing whole brains from months to days. Also, various light microscopy modes were used to demonstrate additional aspects of brain cytoarchitecture such as specific axonal projections and imaging discrete cell populations, including quantitative measurement of their geometric properties. They thoroughly demonstrated proof of principle by reconstructing networks involving cortical, callosal, hippocampal, and neurogenic populations (see Figure 1). Scale promises to revolutionize future light microscopy studies of the brain and possibly other organs!

Figure 1: Three-dimensional reconstruction of yellow fluorescence protein-expressing neurons in Scale-treated mouse brain. The imaging depth is approximately 1.6 mm.