两种荧光示踪剂Fluoro-Gold和Fluoro-Ruby与组织透明技术兼容性研究
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摘要
目的测试两种传统示踪剂Fluoro-Gold(FG)和Fluoro-Ruby(FR)与组织透明技术(3DISCO)的兼容性,并完成立体成像。方法通过切片透明实验评价FG和FR与一种组织透明技术(3DISCO)的兼容性;用FG和FR标记脑和脊髓后,对大鼠全脑和脊髓透明,用光学设备显示标记传导束的三维成像。结果 Fluoro-Gold和Fluoro-Ruby应用3DISCO透明后,信号无衰减,可以完成科研实验要求;对大鼠脑和脊髓组织透明后,应用双光子显微镜观察,可见FG更清晰地展示了神经元及树突,而FR可清晰地显示长距离的轴突。FG与FR表现出了不同的成像结果。结论 Fluoro-Gold和Fluoro-Ruby与3DISCO兼容性较好。通过组织透明技术与光学设备相结合,可以实现组织内部的立体成像。
Objective To test the compatibility of Fluoro-Gold(FG) and Fluoro-Ruby(FR) with tissue clearing technique(3 DISCO)and complete stereoscopic imaging. Methods The compatibility of FG and FR with a tissue clearing technique(3 DISCO) was evaluated by slice experiments. After labeling brain and spinal cord with FG and FR, the three dimensional imaging of labelled conduction bundle was shown by optical equipment and transparent to the whole brain and spinal cord of rats. Results Fluorescence signal of Fluoro-Gold and Fluoro-Ruby after 3 DISCO transparency did not attenuate, and could fulfill the requirements of scientific research. After transparent to the brain and spinal cord of rats, FG resulted in strong retrograde labelling of the neurons and dendrites,while FR resulted in strong retrograde labelling of the long-distance axons under two-photon microscope. Conclusion FluoroGold and Fluoro-Ruby have good compatibility with 3 DISCO. The stereoscopic imaging of tissue can be constructed through the combination of tissue optical clearing techniques and optical equipment.
Objective To test the compatibility of Fluoro-Gold(FG) and Fluoro-Ruby(FR) with tissue clearing technique(3 DISCO)and complete stereoscopic imaging. Methods The compatibility of FG and FR with a tissue clearing technique(3 DISCO) was evaluated by slice experiments. After labeling brain and spinal cord with FG and FR, the three dimensional imaging of labelled conduction bundle was shown by optical equipment and transparent to the whole brain and spinal cord of rats. Results Fluorescence signal of Fluoro-Gold and Fluoro-Ruby after 3 DISCO transparency did not attenuate, and could fulfill the requirements of scientific research. After transparent to the brain and spinal cord of rats, FG resulted in strong retrograde labelling of the neurons and dendrites,while FR resulted in strong retrograde labelling of the long-distance axons under two-photon microscope. Conclusion FluoroGold and Fluoro-Ruby have good compatibility with 3 DISCO. The stereoscopic imaging of tissue can be constructed through the combination of tissue optical clearing techniques and optical equipment.
引文
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2 Garrett WT,McBride RL,Williams JK,Jr.,et al.Fluoro-Gold's toxicity makes it inferior to True Blue for long-term studies of dorsal root ganglion neurons and motoneurons[J].Neurosci Lett,1991,128(1):137-139.
3王培新,张丹,尚爱加,等.组织透明技术[J].神经解剖学杂志,2016,32(1):124-128.
4 Wahl AS,Omlor W,Rubio JC,et al.Neuronal repair.Asynchronous therapy restores motor control by rewiring of the rat corticospinal tract after stroke[J].Science,2014,344(6189):1250-1255.
5 Ren H,Han M,Zhou J,et al.Repair of spinal cord injury by inhibition of astrocyte growth and inflammatory factor synthesis through local delivery of flavopiridol in PLGA nanoparticles[J].Biomaterials,2014,35(24):6585-6594.
6 Spence RD,Kurth F,Itoh N,et al.Bringing CLARITY to gray matter atrophy[J].Neuroimage,2014,101:625-632.
7 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.
8 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.
9 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.
10 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.
11 Hou B,Zhang D,Zhao S,et al.Scalable and DiI-compatible optical clearance of the mammalian brain[J].Front Neuroanat,2015,9:19.
12 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.
13 Kim SY,Chung K,Deisseroth K.Light microscopy mapping of connections in the intact brain[J].Trends Cogn Sci,2013,17(12):596-599.
14张志建,靳森,朱续涛,等.利用嗜神经病毒跨突触追踪神经网络研究进展[J].生命科学,2014,26(6):634-644.
15陶华平.神经系统细胞谱系和神经束路示踪技术[J].杭州师范大学学报:自然科学版,2015,14(4):412-417.
16 Pan C,Cai R,Quacquarelli FP,et al.Shrinkage-mediated imaging of entire organs and organisms using uDISCO[J].Nat Methods,2016,13(10):859-867.
17 Susaki EA,Tainaka K,Perrin D,et al.Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis[J].Cell,2014,157(3):726-739.
18 Liu AKL,Lai HM,Chang RC,et al.Free of acrylamide sodium dodecyl sulphate(SDS)-based tissue clearing(FASTClear):a novel protocol of tissue clearing for three-dimensional visualization of human brain tissues[J].Neuropathol Appl Neurobiol,2017,43(4):346-351.