Chemical imaging of single cells and cell populations using Raman microscopy

点击翻译按钮获取中文摘要

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.**