Titanium dioxide (TiO₂) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO₂ nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ posttreatment labeling approaches to stain TiO₂ nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO₂ nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO₂ nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

中文翻译：

---

TiO 2纳米粒子的细胞内原位标记，用于荧光显微镜检测

生产的二氧化钛（TiO ₂）纳米颗粒具有许多不同的用途，包括开发用于癌症检测和治疗的治疗和诊断纳米颗粒，药物输送，DNA双链断裂的诱导以及特定细胞和亚细胞结构的成像。当前，光学显微镜是生物学和医学病理学最容易获得的成像技术，用于离体检测细胞和组织中的TiO ₂纳米粒子，但其检测限很低，而成像方法则更为敏感（透射电子显微镜，X射线）荧光显微镜等）具有低通量以及技术和操作复杂性。在这里，我们原地描述两个后标记方法可染色细胞吸收的TiO ₂纳米颗粒。第一种方法是利用荧光生物素和链霉抗生物素蛋白在细胞摄取之前和之后标记纳米颗粒。第二种方法是基于铜催化的叠氮化物-炔烃环加成反应，即所谓的“点击化学”，用于标记和检测叠氮化物共轭的TiO ₂纳米颗粒与炔类共轭荧光染料（例如Alexa Fluor 488）一起使用。为了确认这些纳米颗粒的光学荧光信号与Ti元素的分布相匹配，我们在Argonne国家实验室的Advanced Photon Source上使用了同步加速器X射线荧光显微镜（XFM）。钛特异性XFM与共聚焦显微镜检测到的光学荧光位置表现出极好的重叠。因此，使用本文所述方法在原位纳米颗粒标记后，将来使用TiO ₂纳米颗粒进行的实验可能会安全地依赖于共聚焦显微镜。