Chemical imaging of single cells and cell populations using Raman microscopy

使用拉曼显微镜对单细胞和细胞群进行化学成像

• 批准号: RGPIN-2017-04445
• 负责人: Blades, Michael
• 金额: $ 2.04万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711048
• 关键词: Chemical; imaging; single; cells; cell

Raman spectroscopy (RS) is widely used to provide molecular specific information about a sample and is based on the analysis of inelastically scattered light. RS is very useful because it can provide detailed information about the chemical composition and structure of an “analyte”, as well as the chemical and physical environment in which the analyte is imbedded. Among the strengths of RS is its ability to be used to make measurements in aqueous environments, for example biological media under physiological conditions, with no special sample preparation. As a result it is very useful for the study of biochemical systems. RS offers tremendous potential for experimentation involving single cells or small populations, they have traditionally been limited. in terms of in situ analyses, to methods based on standard light microscopy and/or fluorescence image analysis. Light microscopy is limited to cell counting and morphological assessment, and fluorescence provides only a single intensity readout for a given label, whereas RM provides a full vibrational spectrum (chemical signature) at each pixel with no requirement for exogenous labels or added reagents. The one disadvantage of spontaneous RM compared to fluorescence is that it is far less sensitive and thus requires extended exposures in order to obtain good quality images. This proposal will focus primarily on the biological applications of conventional spontaneous Raman and innovative stimulated Raman scattering microscopy (SRSM) system that has been constructed at UBC using funding obtained from the Canada Foundation for Innovation (CFI) and the British Columbia Knowledge Development Fund (BCKDF). This proposed work build on our previous RS research using RS to follow single cells and their progeny through several generations. The previously characterized correlations between observed features in the Raman spectra and loss of pluripotency in stem cells can then be employed at the single cell level and potentially with intracellular resolution allowing comparative analyses of specific cellular compartments. Spatially resolved variations in spectral markers for pluripotency or emerging phenotypes in differentiating cells can then be correlated with readouts from biochemical markers measured using fluorescent probes (e.g., for Oct4, Nanog, SSEA3/4). Combining the capabilities for conventional fluorescence imaging with simultaneous Raman imaging will make it possible to make direct correlations in real time during the initial stages of differentiation of human embryonic stem cells. This work will thus be an important proof-of-principle step towards establishing the potential of spectral markers as indicators of phenotypic changes/variations in other cell types of interest.

拉曼光谱（RS）被广泛用于提供关于样品的分子特异性信息，并且基于非弹性散射光的分析。RS非常有用，因为它可以提供有关“分析物”的化学成分和结构以及分析物嵌入的化学和物理环境的详细信息。 RS的优势之一是它能够用于在水性环境中进行测量，例如生理条件下的生物介质，而无需特殊的样品制备。 因此，它是非常有用的生物化学系统的研究。 RS为涉及单细胞或小群体的实验提供了巨大的潜力，它们传统上受到限制。就原位分析而言，涉及基于标准光学显微镜和/或荧光图像分析的方法。光学显微镜仅限于细胞计数和形态学评估，荧光仅提供给定标记的单一强度读数，而RM在每个像素处提供完整的振动光谱（化学特征），而不需要外源标记或添加试剂。与荧光相比，自发RM的一个缺点是它的灵敏度低得多，因此需要延长曝光时间才能获得高质量的图像。 该提案将主要集中在传统的自发拉曼和创新的受激拉曼散射显微镜（SRSM）系统，已在不列颠哥伦比亚大学使用从加拿大创新基金会（CFI）和不列颠哥伦比亚省知识发展基金（BCKDF）获得的资金建造的生物应用。 这项拟议的工作建立在我们以前的RS研究基础上，使用RS跟踪单细胞及其后代几代。先前表征的拉曼光谱中观察到的特征与干细胞中多能性丧失之间的相关性然后可以在单细胞水平上使用，并且潜在地具有允许对特定细胞区室进行比较分析的细胞内分辨率。然后可以将分化细胞中多能性或新出现的表型的光谱标志物的空间分辨变化与来自使用荧光探针测量的生化标志物的读数（例如，对于Oct 4，Nanog，SSEA 3/4）。将常规荧光成像的能力与同时的拉曼成像相结合，将使得在人胚胎干细胞分化的初始阶段期间在真实的时间内进行直接关联成为可能。因此，这项工作将是建立光谱标记物作为其他感兴趣细胞类型中表型变化/变异指标的潜力的重要原理验证步骤。