Tipping William J, Gould Gwyn W, Faulds Karen, Graham Duncan
Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow G1 1RD, United Kingdom.
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom.
Chem Biomed Imaging. 2025 May 13;3(9):630-635. doi: 10.1021/cbmi.5c00023. eCollection 2025 Sep 22.
Hyperspectral stimulated Raman scattering (SRS) microscopy is rapidly becoming an established method for chemical and biomedical imaging due to the combination of high spatial resolution and chemical information contained within the three-dimensional data set. Chemometric analysis techniques based on linear unmixing, or multivariate analysis, have become indispensable when visualizing hyperspectral data sets. The application of spectral phasor analysis has also been extremely fruitful in this regard, providing a convenient method to retrieve the spatial and chemical components of the data set. Here, we demonstrate the application of spectral phasor analysis for unmixing the overlapping spectral features within the cell-silent region of the SRS spectrum (2000-2300 cm). In doing so, we show it is possible to identify specific Raman signals for DNA, proteins, and lipids following glucose-d metabolism in dividing cells. In addition, we show that spectral phasor analysis is capable of distinguishing different bioorthogonal Raman signals including alkynes and carbon-deuterium (C-D) bonds. We demonstrate the application of spectral phasor analysis for multicomponent unmixing of bioorthogonal Raman groups for high-content cellular imaging applications.
高光谱受激拉曼散射（SRS）显微镜由于其高空间分辨率以及三维数据集中所包含的化学信息，正迅速成为一种成熟的化学和生物医学成像方法。基于线性解混或多变量分析的化学计量学分析技术在可视化高光谱数据集时已变得不可或缺。光谱相量分析在这方面的应用也极其富有成果，它提供了一种便捷的方法来检索数据集的空间和化学成分。在此，我们展示了光谱相量分析在解混SRS光谱（2000 - 2300 cm）细胞沉默区域内重叠光谱特征方面的应用。通过这样做，我们表明在分裂细胞中葡萄糖 - d代谢后，有可能识别出DNA、蛋白质和脂质的特定拉曼信号。此外，我们表明光谱相量分析能够区分不同的生物正交拉曼信号，包括炔烃和碳 - 氘（C - D）键。我们展示了光谱相量分析在生物正交拉曼基团多组分解混用于高内涵细胞成像应用方面的应用。
