组织器官透明化技术在三维成像研究中的应用

doi:10.16098/j.issn.0529-1356.2018.03.022

解剖学报 ›› 2018, Vol. 49 ›› Issue (3): 400-405.doi: 10.16098/j.issn.0529-1356.2018.03.022

组织器官透明化技术在三维成像研究中的应用

李瑛泽邵志华李思光*

同济大学医学院再生医学系，上海 200092

收稿日期:2017-04-14 修回日期:2017-08-28 出版日期:2018-06-06 发布日期:2018-09-18
通讯作者: 李思光 E-mail:siguangli@163.com
基金资助:
国家自然科学基金;国家自然科学基金;国家自然科学基金

Application of clearing methods on tissues in the three-dimensional imaging research

LI Ying-ze SHAO Zhi-hua LI Si-guang*

Department of Regenerative Midicine, School of Medicine, Tongji University, Shanghai 200092, China

Received:2017-04-14 Revised:2017-08-28 Online:2018-06-06 Published:2018-09-18
Contact: LI Si-guang E-mail:siguangli@163.com
Supported by:
Natural Science Foundation of China;Natural Science Foundation of China;Natural Science Foundation of China

摘要/Abstract

摘要：

随着显微成像技术的发展和大数据采集处理系统的不断革新，生物组织成像分析由二维组织切片成像发展到组织器官的三维成像技术。但是，由于组织器官的不透明导致的光线散射，使成像的深度成为一大难题。近年来，组织器官透明化技术取得了很大的发展，该技术通过提高组织的透明度，大大加深了成像深度，从而使得对组织器官的三维成像分析在不需要进行组织切片的情况下得以实现。根据透明化试剂亲水性不同，可将其分为脂溶性透明化技术和水溶性透明化技术，前者减少了组织中的水分，使透明化更加彻底有效；而后者则利用水分子在蛋白分子周围形成的水化膜提高了荧光蛋白稳定性，更利于研究的进行。我们将按照分类对不同透明化技术作出介绍，并从透明化时间长短、荧光保留度等方面进行优劣比较。

关键词: 透明化, 显微成像, 三维重建, 脂质去除, 荧光稳定性

Abstract:

With the development of the microscopy and the big data acquisition processing system, the research direction gradually becomes the construction of biological structures by 3D imaging. However, because of opaque which leads to scattering of imaging light through the tissues, the depth of the imaging becomes a challenge. A technique named “clearing” can increase the organization transparency so that they can enhance the depth of the imaging and have a three-dimensional reconstruction. Being hydrophilic, the clearing reagents were divided into solvent-based clearing and aqueous-based clearing. The solvent-based clearing reduced water in the tissue and aqueous-based clearing used the hydration shell made by water to keep the fluorescent protein stability. In this article we introduced different clearing method and compared them from clearing time and retention in the fluorescence.

Key words: Clearing, Microscopy imaging, Three-dimensional reconstruction, Lipid removal, Fluorescent stability

李瑛泽邵志华李思光. 组织器官透明化技术在三维成像研究中的应用[J]. 解剖学报, 2018, 49(3): 400-405.

LI Ying-ze SHAO Zhi-hua LI Si-guang. Application of clearing methods on tissues in the three-dimensional imaging research[J]. Acta Anatomica Sinica, 2018, 49(3): 400-405.

参考文献

［1］Richardson DS, Lichtman JW. Clarifying tissue clearing［J］. Cell, 2015, 162(2): 246-257.

［2］Micheva KD1, Smith SJ. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits［J］. Neuron, 2007, 55(1): 25-36.

［3］Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy［J］. Science, 1990, 248(4951): 73-76.

［4］Adams MW, Loftus AF, Dunn SE, et al. Light sheet fluorescence microscopy (LSFM) ［J］. Curr Protoc Cytom, 2015, 71: 1-15.

［5］Erturk A, Becker K, Jahrling N，et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO［J］. Nat Protoc, 2012, 7(11): 1983-1995.

［6］Steinke H, Wolff W. A modified Spalteholz technique with preservation of the histology［J］. Ann Anat, 2001, 183(1): 91-95.

［7］Dodt HU, Leischner U, Schierloh A, et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain［J］. Nat Methods, 2007, 4(4): 331-336.

［8］Kolesova H, Capek M, Radochova B, et al. Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts［J］. Histochem Cell Biol, 2016, 146(2): 141-152.

［9］Parra SG, Chia TH, Zinter JP, et al. Multiphoton microscopy of cleared mouse organs［J］. J Biomed Opt, 2010, 15(3): 036017.

［10］Erturk A, Mauch CP, Hellal F, et al. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury［J］. Nat Med, 2011, 18(1): 166-171.

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

［12］Seo J, M Choe, Kim SY. Clearing and labeling techniques for large-scale biological tissues［J］. Mol Cells, 2016, 39(6): 439-446.

［13］Tsai PS, Kaufhold JP, Blinder P, et al. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels［J］. J Neurosci, 2009, 29(46): 14553-14570.

［14］Lin HH, Lai JS, Chin AL,et al. A map of olfactory representation in the drosophila mushroom body［J］. Cell, 2007, 128(6): 1205-1217.

［15］Chiang AS, Lin WY, Liu HP, et al. Insect NMDA receptors mediate juvenile hormone biosynthesis［J］. Proc Natl Acad Sci USA, 2002, 99(1): 37-42.

［16］Moy AJ, Capulong BV, Saager RB, et al. Optical properties of mouse brain tissue after optical clearing with FocusClear［J］. J Biomed Opt, 2015, 20(9): 95010.

［17］Kuwajima T, Sitko AA, Bhansali P, et al. ClearT: a detergent-and solvent-free clearing method for neuronal and non-neuronal tissue［J］. Development, 2013, 140(6): 1364-1368.

［18］Ke MT, Fujimoto S, Imai T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction［J］. Nat Neurosci, 2013, 16(8): 1154-1161.

［19］Ke MT, Imai T. Optical clearing of fixed brain samples using SeeDB［J］. Curr Protoc Neurosci, 2014, 66: Unit 2.22.

［20］Ke MT, Nakai Y, Fujimoto S, et al. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent［J］. Cell Rep, 2016, 14(11): 2718-2732.

［21］Hou B, Zhang D, Zhao S, et al. Scalable and DiI-compatible optical clearance of the mammalian brain［J］. Front Neuroanat, 2015, 9: 19.

［22］Costantini I, Ghobril JP, Di Giovanna AP, et al. A versatile clearing agent for multi-modal brain imaging［J］. Sci Rep, 2015, 5: 9808.

［23］Hama H, Kurokawa H, Kawano H, et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain［J］. Nat Neurosci, 2011, 14(11): 1481-1488.

［24］Hama H, Hioki H, Namiki K, et al. ScaleS: an optical clearing palette for biological imaging［J］. Nat Neurosci, 2015, 18(10): 1518-1529.

［25］Susaki EA, Tainaka K, Perrin D, et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging［J］. Nat Protoc, 2015, 10(11): 1709-1727.

［26］Tainaka K, Kubota SI, Suyama TQ, et al. Whole-body imaging with single-cell resolution by tissue decolorization［J］. Cell, 2014, 159(4): 911-924.

［27］Fumoto S, Nishimura K, Nishida K, et al. Three-dimensional imaging of the intracellular fate of plasmid dna and transgene expression: zsgreen1 and tissue clearing method cubic are an optimal combination for multicolor deep imaging in murine tissues［J］. PLoS One, 2016, 11(1): e0148233.

［28］Tomer R, Ye L, Hsueh B, et al. Advanced CLARITY for rapid and high-resolution imaging of intact tissues［J］. Nat Protoc, 2014, 9(7): 1682-1697.

［29］Epp JR, Niibori Y, Liz Hsiang HL, et al. Optimization of CLARITY for clearing whole-brain and other intact organs(1,2,3) ［J］. JoVE, 2015, 2(3):1-15.

［30］Yang B, Treweek JB, Kulkarni RP, et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing［J］. Cell, 2014, 158(4): 945-958.

［31］Roberts DG, Johnsonbaugh HB, Spence RD, et al. Optical clearing of the mouse central nervous system using passive CLARITY［J］. J Vis Exp, 2016(112):1-9.

[32] Lee E,Choi J,Jo Y,et al.ACT-PRESTO:rapid and consistent tissue clearing and labeling methpd for 3-dimensional (3D)imaging[J].Sci rep,2016,6:18631.

[1]	杨洋史君李琨马渊张少杰侯二飞王超群陈杰王星李志军 . 切除不同范围钩突后颈椎椎间盘的有限元分析[J]. 解剖学报, 2024, 55(1): 88-97.
[2]	米思荣刘广星张镇武于鹏辉鞠晓军饶利兵李莉 . 不同CT层厚下3D打印颅骨的精准度[J]. 解剖学报, 2023, 54(5): 575-581.
[3]	钟铧王颖范应磊任海龙白玛多吉童庆华. 拉萨藏族正常女性数字化骨盆径线测量[J]. 解剖学报, 2023, 54(4): 460-464.
[4]	郭兰前王定宣易刚李焱任杰英 . 基于CT三维重建的喙突形态学分型及其临床意义[J]. 解剖学报, 2023, 54(1): 82-86.
[5]	邓思琪顾玲王恺悦陈虹宇刘宇杰张婕翟效月. 小鼠致密斑后肾小管与入球微动脉毗邻关系的三维可视化[J]. 解剖学报, 2023, 54(1): 87-91.
[6]	张磊李枝青唐小高周鑫李炳坤扶世杰. 载距突解剖形态学分型及特征分析[J]. 解剖学报, 2022, 53(4): 515-519.
[7]	李明波陈晓英郑海霞. 基于 CT 三维重建肩胛骨冈盂切迹的形态学分析及测量[J]. 解剖学报, 2022, 53(3): 335-339.
[8]	张银萍吴菲余慧唐瑶张慧陈伟. 肺内血管的影像解剖学在肺段切除术的临床应用[J]. 解剖学报, 2022, 53(2): 217-224.
[9]	阮彩莲郭宏焘惠雪枫王璐朱慧赵琳刘伯峰李小记. 陕西省陕北地区12~15岁特发性脊柱侧凸与脊柱正常的青少年胸椎椎弓根的形态学分析[J]. 解剖学报, 2022, 53(2): 225-237.
[10]	周安平严杭陈永洪谢东波张玉科郭世涛王建学. 肩峰折角的解剖学分型及临床意义[J]. 解剖学报, 2022, 53(1): 92-95.
[11]	马玉波陈娟翟晓强张同殿种铁王子明赵军 . 附睾管三维重建及解剖[J]. 解剖学报, 2021, 52(6): 960-965.
[12]	丛敬顾玲张婕宋科昕翟效月王效杰. 发生发育小鼠肾近端小管的三维可视化[J]. 解剖学报, 2021, 52(5): 789-794.
[13]	胡定祥李长辉陈亮马晨曦黄河郑瑞清. 基于CT三维重建肩胛下角的形态学分型特点及临床意义[J]. 解剖学报, 2021, 52(5): 767-771.
[14]	武利兵高尚许阳阳王一丹和雨洁王海燕李筱贺李志军. 虚拟仿真手术与常规手术对肾结石治疗效果的比较[J]. 解剖学报, 2021, 52(4): 609-617.
[15]	邬超王一丹关欢欢高明杰张云凤王海燕和雨洁高尚李志军李筱贺. 内蒙古地区枕骨髁及枕骨大孔应用解剖三维数字化测量[J]. 解剖学报, 2021, 52(1): 84-90.

组织器官透明化技术在三维成像研究中的应用

Application of clearing methods on tissues in the three-dimensional imaging research

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价