Abstract
ABSTRACTIntravital microscopy provides dynamic understanding of multiple cell biological processes, but its limited resolution has so far precluded structural analysis. Because it is difficult to capture rare and transient events, only a few attempts have been made to observe specific developmental and pathological processes in animal models using electron microscopy. The multimodal correlative approach that we propose here combines intravital microscopy, microscopic X-ray computed tomography and three-dimensional electron microscopy. It enables a rapid (c.a. 2 weeks) and accurate (<5 µm) correlation of functional imaging to ultrastructural analysis of single cells in a relevant context. We demonstrate the power of our approach by capturing single tumor cells in the vasculature of the cerebral cortex and in subcutaneous tumors, providing unique insights into metastatic events. Providing a significantly improved throughput, our workflow enables multiple sampling, a prerequisite for making correlative imaging a relevant tool to study cell biology in vivo. Owing to the versatility of this workflow, we envision broad applications in various fields of biological research, such as cancer or developmental biology.
Highlights
Metastasis relies on a series of steps, including invasion of the tissue and circulation through the vasculature to reach a secondary distant site (Valastyan and Weinberg, 2011)
Intravital microscopy (IVM) of invasive tumor cells has enabled in vivo studies of the metastatic cascade (Gligorijevic et al, 2014; Kienast et al, 2010)
Intravital microscopy of metastasizing cells in the mouse brain cortex vasculature Correlative imaging of the initial steps of tumor cell extravasation was performed on our established mouse model, in which we could reliably identify fluorescently labeled metastasizing tumor cells and track them over time
Summary
Metastasis relies on a series of steps, including invasion of the tissue and circulation through the vasculature to reach a secondary distant site (Valastyan and Weinberg, 2011). Relevant in vitro models have been created to study these processes (Gligorijevic et al, 2014), they have failed so far to recapitulate the complexity of living tissues. Intravital microscopy (IVM) of invasive tumor cells has enabled in vivo studies of the metastatic cascade (Gligorijevic et al, 2014; Kienast et al, 2010). Tumor progression can be imaged in various animal models upon, for example, orthotopic, subcutaneous or intra-circulation injection of tumor cells (Karreman et al, 2014; Leong et al, 2014; Sahai, 2007; Stoletov et al, 2010).
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