title: Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent.
abstract: Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of >100 μm, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels.
authors.affiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
authors.forename: Meng-Tsen
authors.initials: MT
authors.lastname: Ke
authors.affiliation: Laboratory of Developmental Neurobiology, Brain/Liver Interface Medicine Research Center, Graduate School of Medical Sciences, Kanazawa University, Kanazawa 920-8640, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nakai
authors.affiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujimoto
authors.affiliation: Laboratory of Developmental Neurobiology, Brain/Liver Interface Medicine Research Center, Graduate School of Medical Sciences, Kanazawa University, Kanazawa 920-8640, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Takayama
authors.affiliation: Laboratory for Chromosome Segregation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
authors.forename: Shuhei
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Laboratory for Chromosome Segregation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Tomoya S
authors.initials: TS
authors.lastname: Kitajima
authors.affiliation: Laboratory of Developmental Neurobiology, Brain/Liver Interface Medicine Research Center, Graduate School of Medical Sciences, Kanazawa University, Kanazawa 920-8640, Japan; Mathematical Neuroscience Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-8640, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sato
authors.affiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; PRESTO, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan; Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan. Electronic address: imai@cdb.riken.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imai
mesh_terms: D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism; D001931:Brain Mapping; D007472:Iohexol / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000172:deficiency / Q000235:genetics; D012031:Refractometry; D012503:Saponins / Q000737:chemistry; D013569:Synapses / Q000737:chemistry / Q000378:metabolism
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate; D012503:Saponins; D007472:Iohexol