Chemical imaging of single cells and cell populations using Raman microscopy

拉曼光谱(RS)被广泛用于提供关于样品的分子特定信息，其基础是对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”的化学成分和结构的详细信息，以及分析物所在的化学和物理环境。RS的优点之一是它能够用于在水环境中进行测量，例如在生理条件下的生物介质，而不需要特殊的样品制备。因此，它对生化系统的研究是非常有用的。RS为单细胞或小群体的实验提供了巨大的潜力，但传统上它们都是有限的。在原位分析方面，到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评估，荧光仅为给定标记提供单一强度读数，而RM在每个像素提供完整的振动光谱(化学特征)，不需要外部标记或添加试剂。与荧光相比，自发调光的一个缺点是它的敏感度要低得多，因此需要延长曝光时间才能获得高质量的图像。这项建议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)系统的生物学应用上，该系统是由加拿大创新基金会(CFI)和不列颠哥伦比亚省知识发展基金(BCKDF)资助在不列颠哥伦比亚省建造的。*这项拟议的工作建立在我们之前的RS研究的基础上，使用RS跟踪单细胞及其后代经过几代人。以前在拉曼光谱中观察到的特征与干细胞多能性丧失之间的特征相关性，然后可以在单个细胞水平上使用，并可能与细胞内分辨率相结合，从而允许对特定的细胞隔间进行比较分析。然后，多能性光谱标记或分化细胞中出现的表型的空间分辨变化可以与使用荧光探针(例如，Oct4、Nanog、SSEA3/4)测量的生化标记的读数相关联。将传统的荧光成像能力与同时的拉曼成像能力相结合，将使在人类胚胎干细胞分化的初始阶段进行实时直接关联成为可能。因此，这项工作将是朝着确定光谱标记作为其他感兴趣细胞类型的表型变化/变异指示器的潜力的重要的原则证明步骤。

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.**