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兴标志，但对代谢物定量的新工具的需求至关重要。虽然核磁共振光谱、质谱和拉曼光谱被广泛用于组织提取物或完整组织的分子检测，但这些工具并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满足的需求，从而能够对活肿瘤细胞和完整活检中的代谢物进行定量振动成像。最近开发的SRS显微镜允许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有重叠拉曼带的分子种类。我们建议通过自制调谐放大器阵列实现频谱分散SRS信号的并行检测来克服这一技术障碍。在一项初步研究中，我们展示了活胰腺癌细胞的多重SRS成像，像素停留时间为40秒。在目标1中，我们将开发多重刺激拉曼损失（SRL）显微镜和多变量分析算法，以实现活细胞中脂质代谢物的定量振动成像。在目标2中，我们将开发外延检测的多重SRL显微镜，以实现肿瘤活检的高速，大面积光谱成像。通过完成这两个目标，我们将产生一个高灵敏度，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。该平台将允许在活的癌细胞代谢转化的无标记可视化，这是不可能的蛋白质组学工具。这种能力对于了解肿瘤代谢机制和精确评价靶向肿瘤代谢药物至关重要。该平台还将允许完整肿瘤活检的大面积制图，并提供有关指示癌症侵袭性的代谢生物标志物（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.