A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.
A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.
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DOI：
10.3791/63413
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发表时间：
2022-05-24
影响因子：
1.2
通讯作者：
Ponik, Suzanne M.
中科院分区：
文献类型：
--
作者：
Burkel, Brian M.;Inman, David R.;Virumbrales-Munoz, Maria;Hoffmann, Erica J.;Ponik, Suzanne M.
The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H and Fluorescence Lifetime Imaging Microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse movies of the MMTV-PyMT tumor model to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable. This intravital imaging method utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for more in-depth analysis of 4D intravital movies.
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