Venkateswaran Sharavan Vishaan, Kreuzaler Peter, Maclachlan Catherine, McMahon Greg, Greenidge Gina, Collinson Lucy, Bunch Josephine, Yuneva Mariia
The Francis Crick Institute, London, UK.
University of Cologne, Faculty of Medicine and University Hospital Cologne, Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD), Cologne, Germany.
Nat Protoc. 2025 Jan 29. doi: 10.1038/s41596-024-01118-4.
Tissue microenvironments are extremely complex and heterogeneous. It is challenging to study metabolic interaction between the different cell types in a tissue with the techniques that are currently available. Here we describe a multimodal imaging pipeline that allows cell type identification and nanoscale tracing of stable isotope-labeled compounds. This pipeline extends upon the principles of correlative light, electron and ion microscopy, by combining confocal microscopy reporter or probe-based fluorescence, electron microscopy, stable isotope labeling and nanoscale secondary ion mass spectrometry. We apply this method to murine models of hepatocellular and mammary gland carcinomas to study uptake of glucose derived carbon (C) and glutamine derived nitrogen (N) by tumor-associated immune cells. In vivo labeling with fluorescent-tagged antibodies (B220, CD3, CD8a, CD68) in tandem with confocal microscopy allows for the identification of specific cell types (B cells, T cells and macrophages) in the tumor microenvironment. Subsequent image correlation with electron microscopy offers the contrast and resolution to image membranes and organelles. Nanoscale secondary ion mass spectrometry tracks the enrichment of stable isotopes within these intracellular compartments. The whole protocol described here would take ~6 weeks to perform from start to finish. Our pipeline caters to a broad spectrum of applications as it can easily be adapted to trace the uptake and utilization of any stable isotope-labeled nutrient, drug or a probe by defined cellular populations in any tissue in situ.
组织微环境极其复杂且具有异质性。利用当前可用技术研究组织中不同细胞类型之间的代谢相互作用具有挑战性。在此，我们描述了一种多模态成像流程，该流程能够对稳定同位素标记的化合物进行细胞类型识别和纳米级追踪。该流程在相关光、电子和离子显微镜技术原理的基础上进行了拓展，它结合了共聚焦显微镜报告基因或基于探针的荧光、电子显微镜、稳定同位素标记以及纳米级二次离子质谱技术。我们将此方法应用于肝细胞癌和乳腺癌的小鼠模型，以研究肿瘤相关免疫细胞对葡萄糖衍生碳（C）和谷氨酰胺衍生氮（N）的摄取情况。通过与共聚焦显微镜联用的荧光标记抗体（B220、CD3、CD8a、CD68）进行体内标记，能够识别肿瘤微环境中的特定细胞类型（B细胞、T细胞和巨噬细胞）。随后与电子显微镜进行图像关联，可提供用于成像细胞膜和细胞器的对比度和分辨率。纳米级二次离子质谱技术可追踪这些细胞内区室中稳定同位素的富集情况。此处描述的整个流程从开始到结束大约需要6周时间才能完成。我们的流程适用于广泛的应用，因为它可以很容易地进行调整，以追踪任何稳定同位素标记的营养物质、药物或探针在任何组织原位中特定细胞群体的摄取和利用情况。
