Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland.
Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland; Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Str., 30-387 Krakow, Poland.
Spectrochim Acta A Mol Biomol Spectrosc. 2021 Jul 5;255:119658. doi: 10.1016/j.saa.2021.119658. Epub 2021 Mar 9.
Raman imaging using molecular reporters is a relatively new approach in subcellular investigations. It enables the visualization of organelles in cells with better selectivity and sensitivity compared to the label-free approach. Essentially Raman reporters possess in their structure an alkyne molecular group that can be selectively identified in the spectral region silent for biomolecules, hence facilitate the localization of individual organelles. The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new one, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies. Two-dimensional Raman images were obtained, visualizing the distribution of selected organic compounds in the cell, such as proteins, lipids, and nucleic acids. Additionally, the distribution of EdU, MitoBADY and falcarinol in endothelial cells (ECs) was determined. Moreover, we highlight some drawback of established Raman reporter and the need for testing them in various physiological state of the cell.
基于分子报告物的拉曼成像技术是一种用于亚细胞研究的新兴方法。与非标记方法相比，它能够更有选择性和更灵敏地对细胞器进行可视化。本质上，拉曼报告物在其结构中具有炔基分子基团，该基团可以在对生物分子沉默的光谱区域中被选择性识别，从而有助于个别细胞器的定位。本工作的目的是使用已建立的报告物（EdU 和 MitoBADY）来可视化内皮细胞（HMEC-1）中的主要细胞细胞器，同时还测试了一种新的报告物，即具有亲脂性的法卡林醇。此外，我们还测试了将拉曼报告物作为探针来检测体外模型中内皮功能障碍（ED）诱导后某些细胞器分布变化的可能性。在这两种情况下，诱导的 ED 表现为细胞中形成脂滴，这就是为什么在这些研究中，检测富含脂质的细胞器的良好工具如此重要的原因。获得了二维拉曼图像，可视化了细胞中选定有机化合物（如蛋白质、脂质和核酸）的分布。此外，还确定了 EdU、MitoBADY 和法卡林醇在血管内皮细胞（EC）中的分布。此外，我们强调了一些已建立的拉曼报告物的缺点，并需要在细胞的各种生理状态下对其进行测试。
