Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy.
Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy.
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用于透明组织和超分辨率显微镜的丰富反应实体 (FLARE) 的荧光标记。
DOI：
10.1038/s41596-021-00667-2
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发表时间：
2022
期刊：
影响因子：
14.8
通讯作者：
Vaughan,JoshuaC
中科院分区：
文献类型：
--
作者：
Lee,MinYen;Mao,Chenyi;Glaser,AdamK;Woodworth,MarcusA;Halpern,AaronR;Ali,Adilijiang;Liu,JonathanTC;Vaughan,JoshuaC
荧光显微镜是生物医学研究中的重要工具，但在实现统一或明亮的标记方面面临着相当大的挑战。例如，荧光蛋白仅限于模式生物，抗体结合物可能不一致，难以用于厚标本。为了部分解决这些挑战，我们开发了一种标记方案，可以在薄组织和厚组织或培养细胞中的生物标本上快速显示许多对比良好的关键特征和标志。这种方法使用已建立的反应性荧光团来标记各种生物样本，用于透明组织显微镜或扩展超分辨率显微镜，称为Flare(丰富反应实体的荧光标记)。这些荧光团针对的是化学基团，并揭示了它们在样本上的分布；胺反应的荧光团，如羟基琥珀酰亚胺酯，针对的是蛋白质上可接近的胺，而肼类荧光团针对的是氧化的碳水化合物。由此产生的染色提供的信号类似于传统的常规组织学染色，如H&E或高碘酸席夫，但使用的荧光探针与体积成像兼容。一般来说，Flare的染色是按照碳水化合物、胺和DNA的顺序进行的，根据染色的组合以及生物标本的类型和厚度，这些染色的孵化时间从1小时到1天不等。FLARE功能强大，功能强大，易于在已经进行常规荧光显微镜检查的实验室中实施。
Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid–Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.