Quantitative Spectroscopic Imaging of Cancer Metabolites in Live Cells and Intact

活细胞和完整细胞中癌症代谢物的定量光谱成像

• 批准号: 8622774
• 负责人: Ji-Xin Cheng
• 金额: $ 19.21万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622774
• 关键词: Address; Algorithms; Amino Acids; Amplifiers; Antineoplastic Agents; Area; Beds; Biological Markers; Biopsy; Brain; Cancerous; Carbohydrates; Cells; Chemicals; Cholesterol; Cholesterol Esters; DU145; Dehydration; Detection; Development; Dimethyl Sulfoxide; Drug Targeting; Electron Microscopy; Emulsions; Evaluation; Fatty Acids; Fatty acid glycerol esters; Fingerprint; Fluorescence Microscopy; Frequencies; Genes; Hematoxylin and Eosin Staining Method; Histology; Hybrids; Image; Imagery; Imaging Device; Imaging Techniques; Imaging technology; Kidney; Label; Lasers; Lateral; Life; Lipids; Lipoproteins; Location; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of prostate; Mammary gland; Maps; Mass Spectrum Analysis; Metabolic; Metabolism; Microscope; Microscopy; Molecular; Molecular Analysis; Monitor; Multivariate Analysis; NMR Spectroscopy; Noise; PC3 cell line; Pathogenesis; Patients; Pilot Projects; Play; Procedures; Prognostic Factor; Prostate; Proteins; Proteomics; Pump; Raman Spectrum Analysis; Resolution; Role; Scanning; Signal Transduction; Slice; Slide; Specific qualifier value; Speed; Staging; System; Techniques; Technology; Testing; Time; Tissue Extracts; Tissues; anticancer research; base; cancer cell; human tissue; leukemia; lipid biosynthesis; lipid metabolism; malignant breast neoplasm; malignant mouth neoplasm; neoplastic cell; nutrition; pancreatic cancer cells; prostate cancer cell; public health relevance; quantum; small molecule; spectroscopic imaging; submicron; tool; tumor; tumor metabolism

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.

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，但迫切需要新的代谢物定量工具。尽管核磁共振光谱、质谱和拉曼光谱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 显微镜可以对活细胞中的单个拉曼谱带进行高速、高灵敏度成像。然而，单频 SRS 成像技术的能力有限，因为它无法解析经常具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光谱分散 SRS 信号的并行检测来克服这一技术障碍。在一项试点研究中，我们展示了活体胰腺癌细胞的多重 SRS 成像，像素停留时间为 40 秒。在目标 1 中，我们将开发多重受激拉曼损耗 (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。通过实现这两个目标，我们将生成一个高灵敏度、高速光谱成像平台，用于以亚微米空间分辨率对活细胞进行分子分析。该平台将允许活癌细胞代谢转化的无标记可视化，这是蛋白质组学工具无法实现的。这种能力对于理解癌症代谢的机制和精确评估针对癌症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绘图，并提供有关指示癌症侵袭性的代谢生物标志物（例如胆固醇酯）的信息。