Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy–based connectomics of cellular networks in brain and other tissues.
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We thank H. Sakurai, H. Otsuka and M. Hirano for general assistance, F. Ishidate, B. Zimmermann, R. Wolleschensky, Y. Watanabe, E. Nakasho, H. Kimura, T. Tajima and S. Horie for help with acquiring and analyzing images, RIKEN BSI-Olympus Collaboration Center for technical support, Y. Yoshihara (RIKEN), M. Yamaguchi and K. Mori (The University of Tokyo) for the Nestin promoter–GFP transgenic mice, J.R. Sanes (Harvard) for the YFP-H and GFP-M lines, E. Takahashi (RIKEN) for helpful advice on transgenic mice, S. J. Smith (Stanford) and J.W. Lichtman (Harvard) for helpful advice on tissue clearing, and D. Mou (Harvard), A. Govindarajan, K. Rockland and S. Tonegawa (Massachusetts Institute of Technology), A. Moore and C. Yokoyama (RIKEN) for critical comments. This work was partly supported by grants from Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research on Priority Areas and the Human Frontier Science Program.
Visualizing the 3D architecture of neuronal networks comprised of YFP-expressing neurons in a long quadratic prism (2 mm). A series of X − Y images through the 3D reconstruction data (500 × 500 × 2,000 μm volume) from the cerebral surface to the hippocampus of the YFP-H mouse (13 weeks old). TPEFM with a non-descanned detector and a 20× objective (NA 1.0, WD 2.0 mm) was used.
Visualizing the 3D architecure of neuronal networks comprised of YFP-expressing neurons in a very long quadratic prism (4 mm). A series of X−Y images through the 3D reconstruction data (500 × 500 × 4,000 μm volume) from the cerebral surface to the dentate gyrus of the YFP-H mouse (13 weeks old). TPEFM with a non-descanned detector and a custom designed 25× objective lens (NA 1.0, WD 4.0 mm) was used.
YFP-labeled pyramidal neurons in layers II and III in the right hemisphere and their callosal axons travelling into the left hemisphere. A series of X−Y images through the 3D reconstruction data (10 × 10 × 0.75 mm volume) from anterior to posterior of a brain (10 days old) containing the corpus callosum. A population of layer II/III pyramidal neurons on the right side is highlighted with EYFP fluorescence. A macro zoom confocal microscopy system was used.
Nuclei of proliferating neural stem cells exclusively localized in the subgranular zone in association with a network of blood vessels. Animation (zooming in) of 3D image data (500 × 500 × 1,400 μm volume) in the hippocampal dentate gyrus of a #504 adult (7 weeks old) mouse extensively labeled with Texas Red-labeled lectin. Red, blood vessels; Green, nuclei of proliferating neural stem cells (PNSC) emitting mAG-hGem(1/110) fluorescence. TPEFM with two non-descanned detectors was used.