Quantitative Spectroscopic Imaging of Cancer Metabolites in Live Cells and Intact

描述(由申请人提供)：虽然细胞代谢改变是癌症的一个新的标志，但迫切需要新的工具来量化代谢物。虽然核磁共振光谱、质谱学和拉曼光谱被广泛用于组织提取物或完整组织中的分子检测，但这些工具不能告诉细胞内分析物的空间位置。我们通过开发多重受激拉曼散射(SRS)显微镜来解决这一未得到满足的需求，从而能够对活的肿瘤细胞和完整的活检组织中的代谢物进行定量振动成像。最近发展起来的SRS显微镜可以对活细胞中的单个拉曼波段进行高速、高灵敏度的成像。然而，单频SRS成像技术的能力有限，因为它不能分辨经常具有重叠拉曼光谱的分子物种。我们建议通过并行检测由自制的调谐放大器阵列实现的频谱分散的SRS信号来克服这一技术障碍。在一项初步研究中，我们展示了活体胰腺癌细胞的多重受激拉曼散射成像，像素驻留时间为40°S。在目标1中，我们将发展多重受激拉曼损耗显微镜和多元分析算法，以实现对活体细胞中脂质代谢物的定量振动成像。在目标2中，我们将开发表观检测多路SRL显微镜，以实现对肿瘤活检组织的高速、大面积光谱成像。通过这两个目标的实现，我们将建立一个高灵敏度、高速、光谱成像平台，用于亚微米空间分辨率的活细胞分子分析。该平台将允许对活体癌细胞中的代谢转化进行无标记可视化，这是蛋白质组学工具所不可能做到的。这种能力对于从机制上理解癌症新陈代谢和精确评估针对癌症新陈代谢的药物至关重要。该平台还将允许对完整的肿瘤活检进行大面积测绘，并提供有关指示癌症侵袭性的代谢生物标记物(例如胆固醇酯)的信息。

DESCRIPTION (provided by applicant): While altered cell metabolism is an emerging hallmark of cancer, there is a crucial need of new tools for quantitation of metabolites. Though NMR spectroscopy, mass spectrometry, and Raman spectroscopy are widely used for molecular detection in tissue extracts or intact tissues, these tools do not tell the spatial locations of the analytes inside the cell. We address this unmet need via development of multiplex stimulated Raman scattering (SRS) microscopy to enable quantitative vibrational imaging of metabolites in live tumor cells and intact biopsies. The recently developed SRS microscopy allows high-speed, high-sensitivity imaging of single Raman bands in live cells. However, the single-frequency SRS imaging technique has limited capability because it cannot resolve molecular species that often have overlapped Raman bands. We propose to overcome this technical barrier via parallel detection of spectrally dispersed SRS signals enabled by a homebuilt tuned amplifier array. In a pilot study, we demonstrated multiplex SRS imaging of live pancreatic cancer cells with a pixel dwell time of 40 ¿s. In Aim 1, we will develop multiplex stimulated Raman loss (SRL) microscopy and multivariate analysis algorithm to enable quantitative vibrational imaging of lipid metabolites in live cells. In Aim 2, we will develop epi-detected multiplex SRL microscopy to enable high-speed, large-area spectroscopic imaging of tumor biopsies. By accomplishment of the two aims, we will generate a high- sensitivity, high-speed, spectral imaging platform for molecular analysis of live cells with sub-micron spatial resolution. This platform will permit label-free visualization of metabolic conversion in live cancr cells, which is not possible with proteomics tools. Such capability is critical for mechanistic understanding of cancer metabolism and precise evaluation of drugs targeting cancer metabolism. This platform will also permit large- area mapping of intact tumor biopsies and offer information about metabolic biomarkers (e.g. cholesteryl ester) that are indicative of cancer aggressiveness.