Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow G1 1RD, United Kingdom.
Anal Chem. 2023 May 9;95(18):7244-7253. doi: 10.1021/acs.analchem.3c00212. Epub 2023 Apr 25.
Hyperspectral stimulated Raman scattering (SRS) microscopy is a robust imaging tool for the analysis of biological systems. Here, we present a unique perspective, a label-free spatiotemporal map of mitosis, by integrating hyperspectral SRS microscopy with advanced chemometrics to assess the intrinsic biomolecular properties of an essential process of mammalian life. The application of spectral phasor analysis to multiwavelength SRS images in the high-wavenumber (HWN) region of the Raman spectrum enabled the segmentation of subcellular organelles based on innate SRS spectra. Traditional imaging of DNA is primarily reliant on using fluorescent probes or stains which can affect the biophysical properties of the cell. Here, we demonstrate the label-free visualization of nuclear dynamics during mitosis coupled with an evaluation of its spectral profile in a rapid and reproducible manner. These results provide a snapshot of the cell division cycle and chemical variability between intracellular compartments in single-cell models, which is central to understanding the molecular foundations of these fundamental biological processes. The evaluation of HWN images by phasor analysis also facilitated the differentiation between cells in separate phases of the cell cycle based solely on their nuclear SRS spectral signal, which offers an interesting label-free approach in combination with flow cytometry. Therefore, this study demonstrates that SRS microscopy combined with spectral phasor analysis is a valuable method for detailed optical fingerprinting at the subcellular level.
高光谱受激拉曼散射（SRS）显微镜是一种强大的生物系统分析成像工具。在这里，我们通过将高光谱 SRS 显微镜与先进的化学计量学相结合，提供了一个独特的视角，即无需标记的有丝分裂时空图谱，以评估哺乳动物生命这一基本过程的固有生物分子特性。在拉曼光谱的高波数（HWN）区域中，将光谱相矢量分析应用于多波长 SRS 图像，使我们能够基于固有 SRS 光谱对亚细胞细胞器进行分割。传统的 DNA 成像主要依赖于使用荧光探针或染色剂，这些探针或染色剂会影响细胞的生物物理特性。在这里，我们展示了有丝分裂过程中核动态的无标记可视化，并以快速且可重复的方式评估其光谱特征。这些结果提供了单细胞模型中细胞分裂周期和细胞内隔室之间化学变异性的快照，这对于理解这些基本生物学过程的分子基础至关重要。通过相矢量分析对 HWN 图像的评估还可以仅基于细胞核 SRS 光谱信号来区分细胞周期的不同阶段，这与流式细胞术相结合提供了一种有趣的无标记方法。因此，本研究表明，SRS 显微镜结合光谱相矢量分析是一种在亚细胞水平进行详细光学指纹识别的有价值方法。
