Chemical imaging of single cells and cell populations using Raman microscopy

拉曼光谱（RS）广泛用于提供有关样品的分子特异性信息，并基于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，以及分析物所处的化学和物理环境。RS的优点之一是它能够在水环境中进行测量，例如生理条件下的生物介质，而无需特殊的样品制备。因此，它对生物化学系统的研究非常有用。RS为涉及单细胞或小群体的实验提供了巨大的潜力，这在传统上是有限的。在原位分析方面，采用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，荧光仅为给定标签提供单一强度读数，而RM在每个像素处提供完整的振动谱（化学特征），不需要外源标签或添加试剂。与荧光相比，自发RM的一个缺点是灵敏度低得多，因此需要延长曝光时间才能获得高质量的图像。该提案将主要关注传统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，该系统已在UBC使用加拿大创新基金会（CFI）和不列颠哥伦比亚省知识发展基金（BCKDF）的资助下建立。******这项建议的工作建立在我们以前的RS研究的基础上，使用RS跟踪单细胞及其后代几代。先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相关性可以在单细胞水平上使用，并且可能具有细胞内分辨率，允许对特定细胞区室进行比较分析。分化细胞中多能性或新出现表型的光谱标记的空间分辨差异可以与使用荧光探针测量的生化标记（例如Oct4， Nanog, SSEA3/4）的读数相关联。将常规荧光成像与同步拉曼成像相结合，将有可能在人类胚胎干细胞分化的初始阶段实时进行直接关联。因此，这项工作将是一个重要的原理验证步骤，以建立光谱标记作为其他感兴趣的细胞类型的表型变化/变异指标的潜力

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.**