BRAIN and NERVE－神経研究の進歩73巻10号

2021年10月発行

総説

三次元組織透明化・染色による神経科学研究の現状と未来への展望

著者： 上田泰己¹²

所属機関： ¹東京大学大学院医学系研究科 ²理化学研究所生命機能科学研究センター

ページ範囲：P.1117 - P.1137

文献概要

最先端の組織透明化法は，哺乳類の個々の臓器や体全体のインタクトな組織の細胞解像度の情報を提供する。組織透明化法に光シート顕微鏡による高速撮像と画像解析の自動化とが組み合わさることで，組織検査のコストが削減され，スピードが数桁向上する。さらに，組織透明化の化学は，全臓器の抗体標識を可能にし，厚いヒト組織にも適用可能にする。強力な透明化，標識，イメージング，データ解析を組み合わせることで，科学者たちは，複雑な哺乳類の体や大型のヒト標本の構造的，機能的な細胞情報を加速度的に抽出している。さらに，テラバイト規模のイメージングデータの急速な生成は，大規模データの解析と管理の課題に取り組む効率的な計算アプローチへの高い需要を生み出す。本総説では，組織透明化法が哺乳類の体やヒト標本の偏りのないシステムレベルの俯瞰像をどのようにして提供し得るかを議論し，組織透明化のヒトの神経科学への応用における現在の課題と将来の展望について議論する。

参考文献

1）Spalteholz W: Über das Durchsichtigmachen von Menschlichen und Tierischen Präparaten und Seine Theoretischen Bedingungen, Nebst Anhang: Über Knochenfärbung. S. Hirzel, Leipzig, 1914

2）Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, et al: Structural and molecular interrogation of intact biological systems. Nature 497: 332-337, 2013

3）Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, et al: Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158: 945-958, 2014

4）Renier N, Wu Z, Simon DJ, Yang J, Ariel P, et al: iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159: 896-910, 2014

5）Susaki EA, Tainaka K, Perrin D, Kishino F, Tawara T, et al: Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157: 726-739, 2014

6）Tainaka K, Kubota SI, Suyama TQ, Susaki EA, Perrin D, et al: Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159: 911-924, 2014

7）Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, et al: Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat Med 18: 166-171, 2011

8）Belle M, Godefroy D, Dominici C, Heitz-Marchaland C, Zelina P, et al: A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system. Cell Rep 9: 1191-1201, 2014

9）Treweek JB, Chan KY, Flytzanis NC, Yang B, Deverman BE, et al: Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat Protoc 10: 1860-1896, 2015

10）Costantini I, Ghobril JP, di Giovanna AP, Mascaro ALA, Silvestri L, et al: A versatile clearing agent for multi-modal brain imaging. Sci Rep 5: 9808, 2015　［doi: 10.1038/srep09808］

11）Klingberg A, Hasenberg A, Ludwig-Portugall I, Medyukhina A, Männ L, et al: Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J Am Soc Nephrol 28: 452-459, 2017

12）Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, et al: Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14: 1481-1488, 2011

13）Ke MT, Fujimoto S, Imai T: SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16: 1154-1161, 2013

14）Dodt HU, Leischner U, Schierloh A, Jährling N, Mauch CP, et al: Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods 4: 331-336, 2007

15）Susaki EA, Ueda HR: Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals. Cell Chem Biol 23: 137-157, 2016

16）Tainaka K, Kuno A, Kubota SI, Murakami T, Ueda HR: Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu Rev Cell Dev Biol 32: 713-741, 2016

17）Murakami TC, Mano T, Saikawa S, Horiguchi SA, Shigeta D, et al: A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat Neurosci 21: 625-637, 2018

18）Amiya T, Tanaka T: Phase transitions in crosslinked gels of natural polymers. Macromolecules 20: 1162-1164, 1987

19）Shibayama M, Tanaka T: Volume phase transition and related phenomena of polymer gels. Dušek K (ed): Responsive Gels: Volume Transitions I. Springer, Berlin, 1993, pp1-62

20）Lorentz H: Relation between propagation of light and density of matter. Wied Ann Phys Chem 9: 641-665, 1880

21）Lorenz L: Ueber die Refractionsconstante. Annalen der Physik 247: 70-103, 1880

22）Tainaka K, Murakami TC, Susaki EA, Shimizu C, Saito R, et al: Chemical landscape for tissue clearing based on hydrophilic reagents. Cell Rep 24: 2196-2210, 2018

23）Ueda HR, Dodt HU, Osten P, Economo MN, Chandrashekar J, et al: Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy. Neuron 106: 369-387, 2020

24）Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, et al: Tissue clearing and its applications in neuroscience. Nat Rev Neurosci 21: 61-79, 2020

25）Belle M, Godefroy D, Couly G, Malone SA, Collier F, et al: Tridimensional visualization and analysis of early human development. Cell 169: 161-173, 2017

26）Kim SY, Cho JH, Murray E, Bakh N, Choi H, et al: Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proc Natl Acad Sci U S A 112: E6274-E6283, 2015　［doi: 10.1073/pnas.1510133112］

27）Murray E, Cho JH, Goodwin D, Ku T, Swaney J, et al: Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163: 1500-1514, 2015

28）Renier N, Adams EL, Kirst C, Wu Z, Azevedo R, et al: Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165: 1789-1802, 2016

29）Kubota SI, Takahashi K, Nishida J, Morishita Y, Ehata S, et al: Whole-body profiling of cancer metastasis with single-cell resolution. Cell Rep 20: 236-250, 2017

30）Cai R, Pan C, Ghasemigharagoz A, Todorov MI, Förstera B, et al: Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nat Neurosci 22: 317-327, 2019

31）Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, et al: Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34: 204-209, 2016

32）Bedbrook CN, Deverman BE, Gradinaru V: Viral strategies for targeting the central and peripheral nervous systems. Annu Rev Neurosci 41: 323-348, 2018

33）Keller PJ, Ahrens MB: Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy. Neuron 85: 462-483, 2015

34）Vigouroux RJ, Belle M, Chédotal A: Neuroscience in the third dimension: shedding new light on the brain with tissue clearing. Mol Brain 10: 33, 2017　［doi: 10.1186/s13041-017-0314-y］

35）Ertürk A, Becker K, Jährling N, Mauch CP, Hojer CD, et al: Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc 7: 1983-1995, 2012

36）Liu Z, Gerner MY, van Panhuys N, Levine AG, Rudensky AY, et al: Immune homeostasis enforced by co-localized effector and regulatory T cells. Nature 528: 225-230, 2015

37）Acar M, Kocherlakota KS, Murphy MM, Peyer JG, Oguro H, et al: Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526: 126-130, 2015

38）Espinosa-Medina I, Outin E, Picard CA, Chettouh Z, Dymecki S, et al: Neurodevelopment: parasympathetic ganglia derive from Schwann cell precursors. Science 345: 87-90, 2014

39）Oshimori N, Oristian D, Fuchs E: TGF-β promotes heterogeneity and drug resistance in squamous cell carcinoma. Cell 160: 963-976, 2015

40）Garofalo S, D'Alessandro G, Chece G, Brau F, Maggi L, et al: Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice. Nat Commun 6: 6623, 2015　［doi: 10.1038/ncomms7623］

41）von Neubeck B, Gondi G, Riganti C, Pan C, Parra Damas A, et al: An inhibitory antibody targeting carbonic anhydrase Ⅻ abrogates chemoresistance and significantly reduces lung metastases in an orthotopic breast cancer model in vivo. Int J Cancer 143: 2065-2075, 2018

42）Tanaka N, Kanatani S, Tomer R, Sahlgren C, Kronqvist P, et al: Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Nat Biomed Eng 1: 796-806, 2017

43）Garvalov BK, Ertürk A: Seeing whole-tumour heterogeneity. Nat Biomed Eng 1: 772-774, 2017

44）Pan C, Cai R, Quacquarelli FP, Ghasemigharagoz A, Lourbopoulos A, et al: Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods 13: 859-867, 2016

45）Cai R, Pan C, Ghasemigharagoz A, Todorov MI, Förstera B, et al: Panoptic vDISCO imaging reveals neuronal connectivity, remote trauma effects and meningeal vessels in intact transparent mice. bioRxiv: 374785, 2018　［doi: org/10.1101/374785］

46）Herisson F, Frodermann V, Courties G, Rohde D, Sun Y, et al: Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nat Neurosci 21: 1209-1217, 2018

47）Pan C, Schoppe O, Parra-Damas A, Cai R, Todorov MI, et al: Deep learning reveals cancer metastasis and therapeutic antibody targeting in whole body. bioRxiv: 541862, 2019　［doi: org/10.1101/541862］

48）Bakutkin VV, Maksimova IL, Semyonova TN, Tuchin VV, Kon IL: Controlling optical properties of sclera. Proc SPIE 2393, 1995　［doi: org/10.1117/12.209841］

49）Zimnyakov DA, Tuchin VV, Michin AA, Kon IL, Serov AN: In-vitro human sclera structure analysis using tissue optical immersion effect. Proc SPIE 2673, 1996　［doi: org/10.1117/12.240070］

50）Tuchin VV, Maksimova IL, Zimnyakov DA, Kon IL, Mavlutov AK, et al: Light propagation in tissues with controlled optical properties. Proc SPIE 2925, 1996　［doi: org/10.1117/12.260832］

51）Tuchin VV, Maksimova IL, Zimnyakov DA, Kon IL, Mavlyutov AH, et al: Light propagation in tissues with controlled optical properties. J Biomed Opt 2: 401-417, 1997

52）Bashkatov AN, Tuchin VV, Genina EA, Sinichkin YP, Lakodina NA, et al: Human sclera dynamic spectra: in-vitro and in-vivo measurements. Proc SPIE 3591, 1999　［doi: org/10.1117/12.350592］

53）Tuchin VV, Bashkatov AN, Genina EA, Kochubey V, Lakodina NA, et al: Optics of living tissues with controlled scattering properties. Proc SPIE 3863:10-21, 1999

54）Tuchin VV, Xu X, Wang RK: Dynamic optical coherence tomography in studies of optical clearing, sedimentation, and aggregation of immersed blood. Appl Opt 41: 258-271, 2002

55）Xu X, Wang RK, Elder JB, Tuchin VV: Effect of dextran-induced changes in refractive index and aggregation on optical properties of whole blood. Phys Med Biol 48: 1205-1221, 2003

56）Chance B, Liu H, Kitai T, Zhang Y: Effects of solutes on optical properties of biological materials: models, cells, and tissues. Anal Biochem 227: 351-362, 1995

57）Liu H, Beauvoit B, Kimura M, Chance B: Dependence of tissue optical properties on solute-induced changes in refractive index and osmolarity. J Biomed Opt 1: 200-211, 1996

58）Vargas G, Chan EK, Barton JK, Rylander HG 3rd, Welch AJ: Use of an agent to reduce scattering in skin. Lasers Surg Med 24: 133-141, 1999

59）Vargas G, Chan KF, Thomsen SL, Welch AJ: Use of osmotically active agents to alter optical properties of tissue: effects on the detected fluorescence signal measured through skin. Lasers Surg Med 29: 213-220, 2001

60）Wang RK, Xu X, Tuchin VV, Elder JB: Concurrent enhancement of imaging depth and contrast for optical coherence tomography by hyperosmotic agents. JOSA B 18: 948-953, 2001

61）Xu X, Wang RK: The role of water desorption on optical clearing of biotissue: studied with near infrared reflectance spectroscopy. Med Phys 30: 1246-1253, 2003

62）Jiang J, Wang RK: Comparing the synergistic effects of oleic acid and dimethyl sulfoxide as vehicles for optical clearing of skin tissue in vitro. Phys Med Biol 49: 5283-5294, 2004

63）Choi B, Tsu L, Chen E, Ishak TS, Iskandar SM, et al: Determination of chemical agent optical clearing potential using in vitro human skin. Lasers Surg Med 36: 72-75, 2005

64）Hirshburg J, Choi B, Nelson JS, Yeh AT: Correlation between collagen solubility and skin optical clearing using sugars. Lasers Surg Med 39: 140-144, 2007

65）Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW: 2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microscopy Res Tech 70: 1-9, 2007

66）Aoyagi Y, Kawakami R, Osanai H, Hibi T, Nemoto T: A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain. PLOS ONE 10: e0116280, 2015　［doi: 10.1371/journal.pone.0116280］

67）Kuwajima T, Sitko AA, Bhansali P, Jurgens C, Guido W, et al: ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140: 1364-1368, 2013

68）Tsai PS, Kaufhold JP, Blinder P, Friedman B, Drew PJ, et al: Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J Neurosci 29: 14553-14570, 2009

69）Hou B, Zhang D, Zhao S, Wei M, Yang Z, et al: Scalable and DiI-compatible optical clearance of the mammalian brain. Front Neuroanat 9: 19, 2015　［doi: 10.3389/fnana.2015.00019］

70）Chiang AS, Liu YC, Chiu SL, Hu SH, Huang CY, et al: Three-dimensional mapping of brain neuropils in the cockroach, Diploptera punctata. J Comp Neurol 440: 1-11, 2001

71）Liu YC, Chiang AS: High-resolution confocal imaging and three-dimensional rendering. Methods 30: 86-93, 2003

72）Richardson DS, Lichtman JW: Clarifying tissue clearing. Cell 162: 246-257, 2015

73）Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, et al: ScaleS: an optical clearing palette for biological imaging. Nat Neurosci 18: 1518-1529, 2015

74）Ke MT, Nakai Y, Fujimoto S, Takayama R, Yoshida S, et al: Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Cell Rep 14: 2718-2732, 2016

75）Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, et al: Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10: 1709-1727, 2015

76）Tatsuki F, Sunagawa GA, Shi S, Susaki EA, Yukinaga H, et al: Involvement of Ca(2+)-dependent hyperpolarization in sleep duration in mammals. Neuron 90: 70-85, 2016

77）Economo MN, Clack NG, Lavis LD, Gerfen CR, Svoboda K, et al: A platform for brain-wide imaging and reconstruction of individual neurons. Elife 5: e10566, 2016　［doi: 10.7554/eLife.10566］

78）Wang L, Gillis-Smith S, Peng Y, Zhang J, Chen X, et al: The coding of valence and identity in the mammalian taste system. Nature 558: 127-131, 2018

79）Justus D, Dalügge D, Bothe S, Fuhrmann F, Hannes C, et al: Glutamatergic synaptic integration of locomotion speed via septoentorhinal projections. Nat Neurosci 20: 16-19, 2017

80）Romanov RA, Zeisel A, Bakker J, Girach F, Hellysaz A, et al: Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes. Nat Neurosci 20: 176-188, 2017

81）Lanjakornsiripan D, Pior BJ, Kawaguchi D, Furutachi S, Tahara T, et al: Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers. Nat Commun 9: 1623, 2018　［doi: 10.1038/s41467-018-03940-3］

82）Rousso DL, Qiao M, Kagan RD, Yamagata M, Palmiter RD, et al: Two pairs of ON and OFF retinal ganglion cells are defined by intersectional patterns of transcription factor expression. Cell Rep 15: 1930-1944, 2016

83）Chen JY, Miyanishi M, Wang SK, Yamazaki S, Sinha R, et al: Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature 530: 223-227, 2016

84）Cuccarese MF, Dubach JM, Pfirschke C, Engblom C, Garris C, et al: Heterogeneity of macrophage infiltration and therapeutic response in lung carcinoma revealed by 3D organ imaging. Nat Commun 8: 14293, 2017　［doi: 10.1038/ncomms14293］

85）Davis FM, Lloyd-Lewis B, Harris OB, Kozar S, Winton DJ, et al: Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny. Nat Commun 7: 13053, 2016　［doi: 10.1038/ncomms13053］

86）Li J, Miao L, Shieh D, Spiotto E, Li J, et al: Single-cell lineage tracing reveals that oriented cell division contributes to trabecular morphogenesis and regional specification. Cell Rep 15: 158-170, 2016

87）Yamamoto J, Imai J, Izumi T, Takahashi H, Kawana Y, et al: Neuronal signals regulate obesity induced β-cell proliferation by FoxM1 dependent mechanism. Nat Commun 8: 1930, 2017　［doi: 10.1038/s41467-017-01869-7］

88）Chen F, Tillberg PW, Boyden ES: Optical imaging: expansion microscopy. Science 347: 543-548, 2015

89）Tomer R, Ye L, Hsueh B, Deisseroth K: Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9: 1682-1697, 2014

90）Gradinaru V, Treweek J, Overton K, Deisseroth K: Hydrogel-tissue chemistry: principles and applications. Annu Rev Biophys 47: 355-376, 2018

91）Ku T, Swaney J, Park JY, Albanese A, Murray E, et al: Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat Biotechnol 34: 973-981, 2016

92）Sylwestrak EL, Rajasethupathy P, Wright MA, Jaffe A, Deisseroth K: Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164: 792-804, 2016

93）Park YG, Sohn CH, Chen R, McCue M, Yun DH, et al: Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat Biotechnol 37: 73-83, 2019

94）Renner M, Lancaster MA, Bian S, Choi H, Ku T, et al: Self-organized developmental patterning and differentiation in cerebral organoids. EMBO J 36: 1316-1329, 2017

95）Canter RG, Choi H, Wang J, Watson LA, Yao CG, et al: 3D mapping reveals network-specific amyloid progression and subcortical susceptibility. bioRxiv: 116244, 2017［doi: org/10.1101/116244］

96）Greenbaum A, Chan KY, Dobreva T, Brown D, Balani DH, et al: Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci Transl Med 9: eaah6518, 2017　［doi: 10.1126/scitranslmed.aah6518］

97）Greenbaum A, Jang MJ, Challis C, Gradinaru V: Q＆A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology? BMC Biol 15: 87, 2017　［doi: 10.1186/s12915-017-0421-3］

98）Shah S, Lubeck E, Schwarzkopf M, He TF, Greenbaum A, et al: Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development 143: 2862-2867, 2016

99）DePas WH, Starwalt-Lee R, van Sambeek L, Kumar SR, Gradinaru V, et al: Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling. MBio 7: e00796-e00716, 2016　［doi: 10.1128/mBio.00796-16］

100）Treweek JB, Gradinaru V: Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors. Curr Opin Biotechnol 40: 193-207, 2016

101）Menegas W, Bergan JF, Ogawa SK, Isogai Y, Venkataraju KU, et al: Dopamine neurons projecting to the posterior striatum form an anatomically distinct subclass. Elife 4: e10032, 2015　［doi: 10.7554/eLife.10032］

102）Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, et al: Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci 20: 1172-1179, 2017

103）Liebmann T, Renier N, Bettayeb K, Greengard P, Tessier-Lavigne M, et al: Three-dimensional study of Alzheimer's disease hallmarks using the iDISCO clearing method. Cell Rep 16: 1138-1152, 2016

104）Welniarz Q, Morel MP, Pourchet O, Gallea C, Lamy JC, et al: Non cell-autonomous role of DCC in the guidance of the corticospinal tract at the midline. Sci Rep 7: 410, 2017　［doi: 10.1038/s41598-017-00514-z］

105）Hruska M, Henderson N, le Marchand SJ, Jafri H, Dalva MB: Synaptic nanomodules underlie the organization and plasticity of spine synapses. Nat Neurosci 21: 671-682, 2018

106）Ando K, Laborde Q, Lazar A, Godefroy D, Youssef I, et al: Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D. Acta Neuropathol 128: 457-459, 2014

107）Morawski M, Kirilina E, Scherf N, Jäger C, Reimann K, et al: Developing 3D microscopy with CLARITY on human brain tissue: towards a tool for informing and validating MRI-based histology. Neuroimage 182: 417-428, 2017

108）Phillips J, Laude A, Lightowlers R, Morris CM, Turnbull DM, et al: Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease. Sci Rep 6: 26013, 2016　［doi: 10.1038/srep26013］

109）Liu AKL, Hurry MED, Ng OTW, DeFelice J, Lai HM, et al: Bringing CLARITY to the human brain: visualization of Lewy pathology in three dimensions. Neuropathol Appl Neurobiol 42: 573-587, 2016

110）Lee E, Choi J, Jo Y, Kim JY, Jang YJ, et al: ACT-PRESTO: rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep 6: 18631, 2016　［doi: 10.1038/srep18631］

111）Lai HM, Liu AKL, Ng HHM, Goldfinger MH, Chau TW, et al: Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues. Nat Commun 9: 1066, 2018　［doi: 10.1038/s41467-018-03359-w］

112）Allen JS, Damasio H, Grabowski TJ: Normal neuroanatomical variation in the human brain: an MRI-volumetric study. Am J Phys Anthropol 118: 341-358, 2002

113）Birey F, Andersen J, Makinson CD, Islam S, Wei W, et al: Assembly of functionally integrated human forebrain spheroids. Nature 545: 54-59, 2017

114）Casoni F, Malone SA, Belle M, Luzzati F, Collier F, et al: Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains. Development 143: 3969-3981, 2016

115）Hsueh B, Burns VM, Pauerstein P, Holzem K, Ye L, et al: Pathways to clinical CLARITY: volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease. Sci Rep 7: 5899, 2017　［doi: 10.1038/s41598-017-05614-4］

116）Behjati S, Lindsay S, Teichmann SA, Haniffa M: Mapping human development at single-cell resolution. Development 145: dev152561, 2018　［doi: 10.1242/dev.152561］

117）Dantzer R, O'Connor JC, Freund GG, Johnson RW, Kelley KW: From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 9: 46-56, 2008

118）Kieffer C, Ladinsky MS, Ninh A, Galimidi RP, Bjorkman PJ: Longitudinal imaging of HIV-1 spread in humanized mice with parallel 3D immunofluorescence and electron tomography. Elife 6: e23282, 2017　［doi: 10.7554/eLife.23282］

119）Glaser AK, Reder NP, Chen Y, McCarty EF, Yin C, et al: Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat Biomed Eng 1: 0084, 2017　［doi: 10.1038/s41551-017-0084］

120）Nojima S, Susaki EA, Yoshida K, Takemoto H, Tsujimura N, et al: CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci Rep 7: 9269, 2017　［doi: 10.1038/s41598-017-09117-0］

121）van Royen ME, Verhoef EI, Kweldam CF, van Cappellen WA, Kremers GJ, et al: Three-dimensional microscopic analysis of clinical prostate specimens. Histopathology 69: 985-992, 2016

122）Huisken J, Swoger J, del Bene F, Wittbrodt J, Stelzer EHK: Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305: 1007-1009, 2004

123）Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EHK: Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322: 1065-1069, 2008

124）Voie AH, Burns DH, Spelman FA: Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J Microsc 170: 229-236, 1993

125）Ryan DP, Gould EA, Seedorf GJ, Masihzadeh O, Abman SH, et al: Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy. Nat Commun 8: 612, 2017　［doi: 10.1038/s41467-017-00514-7］

126）Tomer R, Lovett-Barron M, Kauvar I, Andalman A, Burns VM, et al: SPED light sheet microscopy: fast mapping of biological system structure and function. Cell 163: 1796-1806, 2015

127）Gómez-Gaviro MV, Balaban E, Bocancea D, Lorrio MT, Pompeiano M, et al: Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution. Development 144: 2092-2097, 2017

128）Stefaniuk M, Gualda EJ, Pawlowska M, Legutko D, Matryba P, et al: Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene. Sci Rep 6: 28209, 2016　［doi: 10.1038/srep28209］

129）Niedworok CJ, Schwarz I, Ledderose J, Giese G, Conzelmann KK, et al: Charting monosynaptic connectivity maps by two-color light-sheet fluorescence microscopy. Cell Rep 2: 1375-1386, 2012

130）Planchon TA, Gao L, Milkie DE, Davidson MW, Galbraith JA, et al: Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat Methods 8: 417-423, 2011

131）Chen BC, Legant WR, Wang K, Shao L, Milkie DE, et al: Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346: 1257998, 2014　［doi: 10.1126/science.1257998］

132）Wu Y, Wawrzusin P, Senseney J, Fischer RS, Christensen R, et al: Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat Biotechnol 31: 1032-1038, 2013

133）Chhetri RK, Amat F, Wan Y, Höckendorf B, Lemon WC, et al: Whole-animal functional and developmental imaging with isotropic spatial resolution. Nat Methods 12: 1171-1178, 2015

134）Swoger J, Verveer P, Greger K, Huisken J, Stelzer EHK: Multi-view image fusion improves resolution in three-dimensional microscopy. Opt Express 15: 8029-8042, 2007

135）Royer LA, Lemon WC, Chhetri RK, Wan Y, Coleman M, et al: Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat Biotechnol 34: 1267-1278, 2016

136）Royer LA, Lemon WC, Chhetri RK, Keller PJ: A practical guide to adaptive light-sheet microscopy. Nat Protoc 13: 2462-2500, 2018

137）Silvestri L, Müllenbroich MC, Costantini I, di Giovanna AP, Sacconi L, et al: RAPID: real-time image-based autofocus for all wide-field optical microscopy systems. bioRxiv: 170555, 2017　［doi: org/10.1101/170555d］

138）Hörl D, Rusak FR, Preusser F, Tillberg P, Randel N, et al: BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples. Nat Methods 16: 870-874, 2019

139）Gao R, Asano SM, Upadhyayula S, Pisarev I, Milkie DE, et al: Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science 363: eaau8302, 2019　［doi: 10.1126/science.aau8302］

140）Dean KM, Roudot P, Welf ES, Danuser G, Fiolka R: Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys J 108: 2807-2815, 2015

141）Pende M, Becker K, Wanis M, Saghafi S, Kaur R, et al: High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster. Nat Commun 9: 4731, 2018　［doi: 10.1038/s41467-018-07192-z］

142）Amat F, Höckendorf B, Wan Y, Lemon WC, McDole K, et al: Efficient processing and analysis of large-scale light-sheet microscopy data. Nat Protoc 10: 1679-1696, 2015

143）Pietzsch T, Saalfeld S, Preibisch S, Tomancak P: BigDataViewer: visualization and processing for large image data sets. Nat Methods 12: 481-483, 2015

144）Schindelin J, Rueden CT, Hiner MC, Eliceiri KW: The ImageJ ecosystem: an open platform for biomedical image analysis. Mol Reprod Dev 82: 518-529, 2015

145）Pietzsch T, Preibisch S, Tomancák P, Saalfeld S: ImgLib2: generic image processing in Java. Bioinformatics 28: 3009-3011, 2012

146）Preibisch S, Saalfeld S, Schindelin J, Tomancak P: Software for bead-based registration of selective plane illumination microscopy data. Nat Methods 7: 418-419, 2010

147）Preibisch S, Amat F, Stamataki E, Sarov M, Singer RH, et al: Efficient Bayesian-based multiview deconvolution. Nat Methods 11: 645-648, 2014

148）Balázs B, Deschamps J, Albert M, Ries J, Hufnagel L: A real-time compression library for microscopy images. bioRxiv: 164624, 2017　［doi: org/10.1101/164624］

149）Cheeseman BL, Günther U, Gonciarz K, Susik M, Sbalzarini IF: Forget pixels: adaptive particle representation of fluorescence microscopy images. bioRxiv, 2018　［doi: org/10.1101/263061］

150）Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, et al: Fiji: an open-source platform for biological-image analysis. Nat Methods 9: 676-682, 2012

151）Tomer R, Denes AS, Tessmar-Raible K, Arendt D: Profiling by image registration reveals common origin of annelid mushroom bodies and vertebrate pallium. Cell 142: 800-809, 2010

152）Heckscher ES, Long F, Layden MJ, Chuang CH, Manning L, et al: Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience. Development 141: 2524-2532, 2014

153）Ronneberger O, Liu K, Rath M, Rueβ D, Mueller T, et al: ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains. Nat Methods 9: 735-742, 2012

154）Bogovic JA, Hanslovsky P, Wong A, Saalfeld S: Robust registration of calcium images by learned contrast synthesis. 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI), 2016　［doi: 10.1109/ISBI.2016.7493463］

155）Zheng Z, Lauritzen JS, Perlman E, Robinson CG, Nichols M, et al: A complete electron microscopy volume of the brain of adult Drosophila melanogaster. Cell 174: 730-743, 2018

156）Sommer C, Straehle C, Köthe U, Hamprecht FA: Ilastik: interactive learning and segmentation toolkit. 2011 IEEE International Symposium on Biomedical Imaging From Nano to Macro: 230-233, 2011

157）Fürth D, Vaissière T, Tzortzi O, Xuan Y, Märtin A, et al: An interactive framework for whole-brain maps at cellular resolution. Nat Neurosci 21: 139-149, 2018

158）Oh SW, Harris JA, Ng L, Winslow B, Cain N, et al: A mesoscale connectome of the mouse brain. Nature 508: 207-214, 2014

159）Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, et al: Genome-wide atlas of gene expression in the adult mouse brain. Nature 445: 168-176, 2007

160）Saalfeld S, Cardona A, Hartenstein V, Tomancak P: CATMAID: collaborative annotation toolkit for massive amounts of image data. Bioinformatics 25: 1984-1986, 2009

161）Schneider-Mizell CM, Gerhard S, Longair M, Kazimiers T, Li F, et al: Quantitative neuroanatomy for connectomics in Drosophila. Elife 5: e12059, 2016　［doi: 10.7554/eLife.12059］

162）Amat F, Lemon W, Mossing DP, McDole K, Wan Y, et al: Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data. Nat Methods 11: 951-958, 2014

163）Bria A, Iannello G: TeraStitcher: a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images. BMC Bioinformatics 13: 316, 2012　［doi: 10.1186/1471-2105-13-316］

164）Wolff C, Tinevez JY, Pietzsch T, Stamataki E, Harich B, et al: Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking. bioRxiv: 112623, 2017　［doi: org/10.1101/112623］

165）Moffitt JR, Bambah-Mukku D, Eichhorn SW, Vaughn E, Shekhar K, et al: Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region. Science 362: eaau5324, 2018　［doi: 10.1126/science.aau5324］

166）Susaki EA, Shimizu C, Kuno A, Tainaka K, Li X, et al: Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues. Nat Commun 11: 1982, 2020　［doi: 10.1038/s41467-020-15906-5］

167）Lorentz HA: Ueber die beziehung zwischen der fortpflanzungsgeschwindigkeit des lichtes und der körperdichte. Annalen der Physik 245: 641-665, 1880

168）Matsumoto K, Mitani TT, Horiguchi SA, Kaneshiro J, Murakami TC, et al: Advanced CUBIC tissue clearing for whole-organ cell profiling. Nat Protoc 14: 3506-3537, 2019