High-resolution volumetric imaging of metabolic activity in tissues and its application to tumor metabolism

项目摘要 遗传和新陈代谢是生命的两个决定性特征。了解动物的新陈代谢 这对解开健康和疾病中许多生物过程的机械基础至关重要， 发育、衰老和癌症。对于正常生理学来说，它是合成、转化和降解的过程。 生物分子（即，代谢活动），执行动物的遗传蓝图。对于疾病，代谢 重编程是癌症的一个标志（如瓦尔堡效应）。然而，流行的成像技术 例如磁共振光谱学、正电子发射断层摄影术、成像质谱学， 荧光显微术对于非侵入性高分辨率代谢成像都表现出固有的局限性。 因此，目前还没有代谢成像技术可以提供所需的组合， 原位探测、单细胞分辨率和三维（3D）组织的体积成像。等 技术将有助于对正常生理和疾病进展的机械理解， 肿瘤。本项目的目标是开发一种新的光学技术，用于原位高分辨率体积测量。 动物组织中代谢活动的成像，捕捉整个3D组织中每个细胞的代谢状态。 我们已经制定了一个多学科的方法，包括探索在体内标记探针，新的样品， 治疗方案，新的显微镜仪器建设，和多变量计算分析。 目的1是开发重水（D2O）作为与新兴的受激耦合的通用探针 拉曼散射（SRS）显微镜，以多重方式监测组织的代谢活动。我们 初步数据已证明其可行性。高级计算分析和超光谱SRS 将开发仪器以实现全面的代谢分析。目标2旨在建立一个 体积成像方法，以生成深入组织的3D代谢活动图。我们建议发展 拉曼定制的清除配方，将打开厚组织和整个器官的体积SRS成像。 我们的初步数据已经实现了超过10倍的SRS成像深度扩展比以前可能的。 目标3将整合和调整目标1和目标2的技术发展，用于代谢成像 肿瘤组织的异质性。我们建议建立相关荧光和SRS显微镜， 使用多变量计算分析。这些发展将被整合，以获得细胞类型特异性 3D肿瘤组织内的代谢活性分析（包括不同类型的新合成分子）， 有助于了解癌症的原因、进展和完善的治疗策略。 1

Project Summary Genetics and metabolism are two defining characteristics of life. Understanding metabolism in animals is critical to unraveling the mechanistic basis of many biological processes in health and diseases, such as development, aging and cancer. For normal physiology, it is the synthesis, transformation and degradation of biomolecules (i.e., metabolic activity) that carry out the genetic blueprint of animals. For diseases, metabolic reprogramming is a hallmark for cancer (such as the Warburg effect). However, popular imaging techniques such as magnetic resonance spectroscopy, positron emission tomography, imaging mass spectroscopy and fluorescence microscopy all exhibit inherent limitations towards noninvasive high-resolution metabolic imaging. Therefore, there is no current metabolic imaging technique that can offer the desired combination of in situ probing, single-cell resolution, and volumetric imaging of three-dimensional (3D) tissues. Such technology would contribute to mechanistic understanding of normal physiology and disease progression such as tumor. The goal of this project is to develop a novel optical technology for in situ high-resolution volumetric imaging of metabolic activity in animal tissues, capturing metabolic status of every cell throughout 3D tissues. We have laid out a multi-disciplinary approach including exploration of in vivo labeling probe, novel sample treatment protocol, new microscope instrumentation construction, and multivariate computational analysis. Aim 1 is about developing heavy water (D2O) as a universal probe to be coupled with the emerging stimulated Raman scattering (SRS) microscopy, to monitor metabolic activities of tissues in a multiplex manner. Our preliminary data have demonstrated the feasibility. Advanced computational analysis and hyperspectral SRS instrumentation will be developed to achieve comprehensive metabolic profiling. Aim 2 aims to establish a volumetric imaging method to generate 3D metabolic activity maps deep into tissues. We propose to develop Raman-tailored clearing recipe that will open up volumetric SRS imaging of thick tissues and whole organs. Our preliminary data have achieved more than 10 times SRS imaging depth extension than previously possible. Aim 3 will integrate and tailor the technical development from Aim 1 and Aim 2 for imaging metabolic heterogeneity of tumor tissue. We propose to construct correlative fluorescence and SRS microscope and employ multivariate computational analysis. These development will be integrated to obtain cell-type-specific metabolic activity profiling (including different types of newly-synthesized molecules) within 3D tumor tissues, facilitating understanding of the causes, progression and refined treatment strategies of cancer. 1