Bentolila Nathan Y, Barnhill Raymond L, Lugassy Claire, Bentolila Laurent A
Genesis Innovation Lab, YULA School, Los Angeles, CA, USA.
Department of Pathology, Institut Curie, University of Paris René Descartes, Paris, France.
Methods Mol Biol. 2018;1755:223-232. doi: 10.1007/978-1-4939-7724-6_15.
Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.
通过双光子激发（2PE）激光扫描显微镜对报告基因表达进行无创成像，非常适合在深部组织的体内进行动态和多维成像，直至单细胞检测灵敏度。在这里，我们使用2PE显微镜将绿色荧光蛋白（GFP）可视化为植入小鼠耳皮肤真皮层的人黑色素瘤细胞中的报告基因。我们首先提供一种逐步的方法来建立小鼠耳部皮肤的2PE成像模型，然后将其应用于体内原发性肿瘤及其相关脉管系统的观察。这种方法微创，允许随时间重复成像，并对完整动物体内的恶性生长进行连续视觉监测。通过2PE对小型活体动物中的荧光报告基因表达进行成像，提供了一种独特的工具，可在体内以高空间和时间分辨率研究癌症生物学中的关键途径和分子事件，如肿瘤发生和转移。
