A comprehensive understanding of interactions between nanoparticles (NPs) and biological components is critical to the clinical application of NPs and nanomedicine. Here we provide a step-by-step correlative imaging approach to investigate plasmonic NPs of different aggregation states at the single-cell level. Traceable spherical nucleic acids (SNAs) are fabricated by decorating 50-nm spherical gold NPs with fluorophore-labeled DNA, serving as dually emissive (fluorescent and plasmonic) NPs. The in situ correlative imaging with dark-field microscopy (DFM) and fluorescence microscopy (FM) reveals intracellular distribution of SNAs, whereas DFM combined with scanning electron microscopy (SEM) allows semi-quantification of SNA clustering states in solution. The imaging data are analyzed by ImageJ and a colorimetry-based algorithm written in Python. The clustering states of SNAs in a single cell can be efficiently distinguished within 20 s. This method can be readily installed to monitor real-time endocytosis and cellular distribution of plasmonic NPs of different aggregation states and to quantitatively image targets of interest (e.g., specific DNA, messenger RNA, peptides or proteins) in living cells. The entire procedure can be completed in 3–5 d and requires standard DFM, FM and SEM imaging and data analysis skills and equipment.

全面了解纳米粒子 (NPs) 与生物成分之间的相互作用对于 NPs 和纳米药物的临床应用至关重要。在这里，我们提供了一种分步相关成像方法来研究单细胞水平上不同聚集状态的等离子体 NP。可追踪球形核酸 (SNA) 是通过用荧光团标记的 DNA 装饰 50 nm 球形金 NPs 制成的，用作双重发射（荧光和等离子体） NPs。暗场显微镜 (DFM) 和荧光显微镜 (FM) 的原位相关成像揭示了 SNA 的细胞内分布，而 DFM 与扫描电子显微镜 (SEM) 相结合可以半量化溶液中的 SNA 聚类状态。成像数据由 ImageJ 和用 Python 编写的基于比色法的算法进行分析。单个细胞中 SNA 的聚类状态可以在 20 秒内有效区分。该方法可以很容易地安装以监测不同聚集状态的等离子体 NP 的实时内吞作用和细胞分布，并定量成像活细胞中感兴趣的目标（例如，特定的 DNA、信使 RNA、肽或蛋白质）。整个过程可在 3-5 天内完成，需要标准的 DFM、FM 和 SEM 成像和数据分析技能和设备。