Intravital fluorescence microscopy with negative contrast.
Intravital fluorescence microscopy with negative contrast.
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DOI：
10.1371/journal.pone.0255204
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Lin CP
中科院分区：
文献类型：
--
作者：
Wu JW;Jung Y;Yeh SA;Seo Y;Runnels JM;Burns CS;Mizoguchi T;Ito K;Spencer JA;Lin CP
活体显微镜(IVM)的发展使得在最小扰动的情况下研究完整生物的自然微环境中的细胞组织和动力学成为可能。追踪特定细胞群体并监测其相互作用的能力为在活体内可视化细胞生物学开辟了新的视野，然而标准的荧光细胞标记方法的成功伴随着“阴暗面”，即未标记的细胞是看不见的，使标记的细胞或结构看起来孤立在空间中，与周围环境无关，缺乏适当的生物学背景。在这里，我们描述了一种新的方法，通过利用细胞外(间质)液体的无处不在及其容易被常用的血管和淋巴示踪剂进行荧光标记来“填充空洞”。我们发现，在IVM的血管和淋巴管的常规标记过程中，常用的荧光示踪剂很容易进入骨髓(BM)和淋巴结(LN)的间质间隙，勾勒出未标记的细胞并形成负对比图像，补充标准的(阳性)细胞标记方法。该方法简单而强大，提供了细胞景观的全面视图，如细胞密度和空间分布，以及动态过程，如细胞运动和跨血管内皮细胞的迁移。染料的细胞外定位和间质流动为延长活体内时间推移成像和光漂白提供了有利条件，毒性最小。
Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a “dark side” in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for “filling in the void” by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.
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影响因子：
1.2
作者：
Drew PJ;Blinder P;Cauwenberghs G;Shih AY;Kleinfeld D
通讯作者：
Kleinfeld D
DOI：
10.1126/science.abb8739
发表时间：
2020-10-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Nedergaard M;Goldman SA
通讯作者：
Goldman SA
DOI：
10.1084/jem.192.10.1425
发表时间：
2000-11-20
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Gretz JE;Norbury CC;Anderson AO;Proudfoot AE;Shaw S
通讯作者：
Shaw S
影响因子：
3.7
作者：
Kim TN;Goodwill PW;Chen Y;Conolly SM;Schaffer CB;Liepmann D;Wang RA
通讯作者：
Wang RA
影响因子：
16.6
作者：
Koechlein CS;Harris JR;Lee TK;Weeks J;Fox RG;Zimdahl B;Ito T;Blevins A;Jung SH;Chute JP;Chourasia A;Covert MW;Reya T
通讯作者：
Reya T