了解纳米药物的细胞内行为以及受纳米材料-生物学（纳米-生物）相互作用影响的亚细胞结构的形态变化，有助于指导用于临床转化的纳米药物的安全设计、制造和评估。在纳米尺度上对完整单细胞中的纳米生物相互作用进行原位和无标记分析仍然具有挑战性。我们开发了一种基于 X 射线显微镜的方法，无需以纳米分辨率进行标记即可直接可视化 2D 或 3D 细胞内分布，并原位分析纳米药物的化学转化。在这里，我们描述了细胞样品制备、光束线选择、数据采集和分析的优化工作流程。我们以几种模型生物纳米材料为例，用软、硬X射线分析了纳米药物在各种原代血细胞、巨噬细胞、树突状细胞、单核细胞和癌细胞中的定位，以及一些细胞器的形态。我们的协议已在三个光束线设施上成功实施：北京同步辐射装置4W1A、上海同步辐射装置BL08U1A和国家同步辐射实验室BL07W。该协议可以在约 2-5 天内完成，具体取决于细胞类型、纳米材料的孵育时间和所选的 X 射线束线。该协议能够通过使用这个通用且强大的平台，对各种含金属纳米材料进行原位分析，细胞内吞作用、分布和排泄以及受细胞系或原代细胞中纳米药物影响的相应亚细胞形态变化的可视化。结果有助于理解纳米生物相互作用的真正原理和机制。© 2023。施普林格自然有限公司。

Understanding the intracellular behaviors of nanomedicines and morphology variation of subcellular architecture impacted by nanomaterial-biology (nano-bio) interactions could help guide the safe-by-design, manufacturing and evaluation of nanomedicines for clinical translation. The in situ and label-free analysis of nano-bio interactions in intact single cells at nanoscale remains challenging. We developed an approach based on X-ray microscopy to directly visualize the 2D or 3D intracellular distribution without labeling at nanometer resolution and analyze the chemical transformation of nanomedicines in situ. Here, we describe an optimized workflow for cell sample preparation, beamline selection, data acquisition and analysis. With several model bionanomaterials as examples, we analyze the localization of nanomedicines in various primary blood cells, macrophages, dendritic cells, monocytes and cancer cells, as well as the morphology of some organelles with soft and hard X-rays. Our protocol has been successfully implemented at three beamline facilities: 4W1A of Beijing Synchrotron Radiation Facility, BL08U1A of Shanghai Synchrotron Radiation Facility and BL07W of the National Synchrotron Radiation Laboratory. This protocol can be completed in ~2-5 d, depending on the cell types, their incubation times with nanomaterials and the selected X-ray beamline. The protocol enables the in situ analysis of the varieties of metal-containing nanomaterials, visualization of intracellular endocytosis, distribution and excretion and corresponding subcellular morphological variation influenced by nanomedicines in cell lines or primary cells by using this universal and robust platform. The results facilitate the understanding of the true principle and mechanism underlying the nano-bio interaction.© 2023. Springer Nature Limited.