Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, 24 Karłowicza Street, 85-092 Bydgoszcz, Poland.
Acta Histochem. 2013 Jun;115(5):487-95. doi: 10.1016/j.acthis.2012.11.005. Epub 2013 Jan 9.
Quantum dots (QDs) are fluorescent nanocrystals whose unique properties are fundamentally different from organic fluorophores. Moreover, their cores display sufficient electron density to be visible under transmission electron microscopy (TEM). Here, we report a technique for phalloidin-based TEM detection of F-actin. The ultrastructural reorganization of F-actin after arsenic trioxide (ATO) treatment was estimated using a combination of pre- and post-embedding techniques with biotinylated phalloidin and QD-streptavidin conjugates or colloidal gold (AU) conjugated to streptavidin. Ultrastructural studies showed ATO-induced apoptosis of HL-60 cells. Moreover, different patterns of QD-labeled F-actin after ATO treatment were seen. In the case of AU labeling, only a few gold particles were seen and it was impossible to see any difference in F-actin distribution. TEM imaging experiments using QDs and colloidal gold (AU) showed that the strategy of bioconjugation of nanoprobes is the most important factor in biotinylated phalloidin detection of F-actin using streptavidin-coated nanoparticles, especially at the ultrastructural level. Additionally, the results presented in present study confirm the essential role of F-actin in chromatin reorganization during cell death processes.
量子点 (QDs) 是荧光纳米晶体，其独特的性质与有机荧光团根本不同。此外，它们的核心具有足够的电子密度，在透射电子显微镜 (TEM) 下可见。在这里，我们报告了一种基于鬼笔环肽的 TEM 检测 F-肌动蛋白的技术。使用生物素化鬼笔环肽和 QD-链霉亲和素或胶体金 (AU) 与链霉亲和素缀合的预包埋和后包埋技术的组合，估计了三氧化二砷 (ATO) 处理后 F-肌动蛋白的超微结构重排。超微结构研究表明 ATO 诱导 HL-60 细胞凋亡。此外，还观察到 ATO 处理后 QD 标记的 F-肌动蛋白的不同模式。在用 AU 标记的情况下，只看到少数金颗粒，无法看到 F-肌动蛋白分布的任何差异。使用 QD 和胶体金 (AU) 的 TEM 成像实验表明，纳米探针的生物共轭策略是使用链霉亲和素包被的纳米颗粒检测 F-肌动蛋白的生物素化鬼笔环肽的最重要因素，特别是在超微结构水平上。此外，本研究的结果证实了 F-肌动蛋白在细胞死亡过程中染色质重排中的重要作用。
