Nanolab Research Centre, Focas Research Institute, Dublin Institute of Technology (DIT), Camden Row, Dublin, 8, Ireland.
Analyst. 2012 Mar 7;137(5):1111-9. doi: 10.1039/c2an15977e. Epub 2012 Jan 25.
Raman microscopy is employed to spectroscopically image biological cells previously exposed to fluorescently labelled polystyrene nanoparticles and, in combination with K-means clustering and principal component analysis (PCA), is demonstrated to be capable of localising the nanoparticles and identifying the subcellular environment based on the molecular spectroscopic signatures. The neutral nanoparticles of 50 nm or 100 nm, as characterised by dynamic light scattering, are shown to be non-toxic to a human lung adenocarcinoma cell-line (A549), according to a range of cytotoxicity assays including Neutral Red, Alamar Blue, Coomassie Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal fluorescence microscopy identifies intracellular fluorescence due to the nanoparticle exposure, but the fluorescence distribution is spatially diffuse, potentially due to detachment of the dye from the nanoparticles, and the technique fails to unambiguously identify the distribution of the nanoparticles within the cells. Raman spectroscopic mapping of the cells in combination with K-means cluster analysis is used to clearly identify and localise the polystyrene nanoparticles in exposed cells, based on their characteristic spectroscopic signatures. PCA identifies the local environment as rich in lipidic signatures which are associated with localisation of the nanoparticles in the endoplasmic reticulum. The importance of optimised cell growth conditions and fixation processes is highlighted. The preliminary study demonstrates the potential of the technique to unambiguously identify and locate nonfluorescent nanoparticles in cells and to probe not only the local environment but also changes in the cell metabolism which may be associated with cytotoxic responses.
拉曼显微镜用于对先前暴露于荧光标记聚苯乙烯纳米粒子的生物细胞进行光谱成像，并且结合 K-均值聚类和主成分分析 (PCA)，被证明能够基于分子光谱特征对纳米粒子进行定位并识别亚细胞环境。通过动态光散射表征的 50nm 或 100nm 中性纳米粒子，根据一系列细胞毒性测定，包括中性红、阿马尔蓝、考马斯亮蓝和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐 (MTT)，被证明对人肺腺癌细胞系 (A549) 没有毒性。共聚焦荧光显微镜识别出由于纳米粒子暴露而导致的细胞内荧光，但荧光分布是空间弥散的，可能是由于染料从纳米粒子上脱落，并且该技术无法明确识别纳米粒子在细胞内的分布。结合 K-均值聚类分析的细胞拉曼光谱映射用于基于其特征光谱特征，明确识别和定位暴露细胞中的聚苯乙烯纳米粒子。PCA 确定了富含脂质特征的局部环境，这些特征与纳米粒子在内质网中的定位有关。优化细胞生长条件和固定过程的重要性得到了强调。初步研究表明，该技术具有明确识别和定位细胞中非荧光纳米粒子的潜力，并不仅可以探测局部环境，还可以探测与细胞毒性反应相关的细胞代谢变化。
