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Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

机译：使用解剖参考点将活体内多光子显微镜与侵袭性肿瘤细胞的3D电子显微镜相关

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Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.
机译：关联显微镜结合了光学和电子显微镜的优点，可以对稀有事件和瞬态事件进行高分辨率成像。在复杂而庞大的样本（例如整个活生物体）中进行相关显微镜检查是一项耗时且容易出错的任务。在这里，我们研究了相关的方法，这些方法依赖于使用样品的人工和内源性结构特征作为与活体荧光显微镜和电子显微镜相关的参考点。为了以超微结构的准确性研究体内肿瘤细胞的行为，需要一种可靠的方法来检索在组织内深处成像的单个肿瘤细胞。为此，将经荧光标记的肿瘤细胞皮下注射到小鼠的耳朵中，并使用双光子激发显微镜成像。使用近红外烙印，可以在皮肤水平上标记样品中成像区域的位置，以便对其进行精确的采集。在准备好用于电子显微镜的样品后，人工烙印和解剖标志的共同使用可以在连续切片标本的同时靶向和接近目标细胞。我们在这里描述了三个过程，这些过程显示了如何利用组织中的结构特征的三维（3D）映射来精确地关联两个成像模态之间的关系，而不必依赖于使用人工引入的感兴趣区域的标记。此处采用的方法促进了浸润性肿瘤细胞的活体内成像和纳米成像之间的联系，从而在研究肿瘤浸润和转移时使功能与结构相关。

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