Martinić Ivana, Eliseeva Svetlana V, Nguyen Tu N, Foucher Frédéric, Gosset David, Westall Frances, Pecoraro Vincent L, Petoud Stéphane
Centre de Biophysique Moléculaire , CNRS , UPR 4301 , 45071 Orléans Cedex 2 , France . Email:
Department of Chemistry , Willard H. Dow Laboratories , University of Michigan , 930 N. University Ave , Michigan 48109 , USA . Email:
Chem Sci. 2017 Sep 1;8(9):6042-6050. doi: 10.1039/c7sc01872j. Epub 2017 Aug 8.
Cell fixation is an essential approach for preserving cell morphology, allowing the targeting and labelling of biomolecules with fluorescent probes. One of the key requirements for more efficient fluorescent labelling is the preservation of cell morphology, which usually requires a combination of several fixation techniques. In addition, the use of a counter stain is often essential to improve the contrast of the fluorescent probes. Current agents possess significant limitations, such as low resistance toward photobleaching and sensitivity to changes in the microenvironment. Luminescent Ln 'encapsulated sandwich' metallacrowns (MCs) overcome these drawbacks and offer complementary advantages. In particular, they emit sharp emission bands, possess a large difference between excitation and emission wavelengths and do not photobleach. Herein, MCs formed with pyrazinehydroxamic acid (Ln[Zn(ii)MC], Ln = Yb, Nd) were used, combined with near-infrared (NIR) counter staining and fixation agents for HeLa cells upon an initial five minute exposure to UV-A light. The validity and quality of the cell fixation were assessed with Raman spectroscopy. Analysis of the NIR luminescence properties of these MCs was performed under different experimental conditions, including in a suspension of stained cells. Moreover, the high emission intensity of Ln[Zn(ii)MC] in the NIR region allows these MCs to be used for imaging with standard CCD cameras installed on routine fluorescence microscopes. Finally, the NIR-emitting Ln[Zn(ii)MC] compounds combine, within a single molecule, features such as cell fixation and staining abilities, good photostability and minimal sensitivity of the emission bands to the local microenvironment, and they are highly promising for establishing the next generation of imaging agents with a single biodistribution.
细胞固定是保存细胞形态的重要方法，可实现用荧光探针靶向和标记生物分子。更高效荧光标记的关键要求之一是保持细胞形态，这通常需要多种固定技术相结合。此外，使用复染剂通常对于提高荧光探针的对比度至关重要。目前的试剂存在显著局限性，如对光漂白的抗性低以及对微环境变化敏感。发光的镧系元素“封装夹心”金属冠醚（MCs）克服了这些缺点并具有互补优势。特别是，它们发射尖锐的发射带，激发和发射波长之间存在很大差异且不会发生光漂白。在此，使用由吡嗪异羟肟酸形成的MCs（Ln[Zn(ii)MC]，Ln = Yb、Nd），在最初暴露于UV - A光五分钟后，与近红外（NIR）复染剂和固定剂一起用于HeLa细胞。用拉曼光谱评估细胞固定的有效性和质量。在不同实验条件下，包括在染色细胞悬液中，对这些MCs的近红外发光特性进行了分析。此外，Ln[Zn(ii)MC]在近红外区域的高发射强度使得这些MCs可用于安装在常规荧光显微镜上的标准电荷耦合器件（CCD）相机成像。最后，发射近红外光 的Ln[Zn(ii)MC]化合物在单个分子内结合了细胞固定和染色能力、良好的光稳定性以及发射带对局部微环境的最小敏感性等特性，它们在建立具有单一生物分布的下一代成像剂方面极具前景。
