SUPA, School of Physics and Astronomy, University of St Andrews, North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
Anal Chem. 2010 Jan 15;82(2):738-45. doi: 10.1021/ac9026737.
Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures.
无标记的单细胞化学特征分析是生物医学研究的一个重要目标。标准的拉曼光谱为非侵入性分析生物样本提供了内在的生化标志物，但常常受到荧光背景的干扰。在本文中，我们提出了一种创新的调制拉曼光谱技术，以从荧光背景中滤除拉曼光谱。该方法基于这样一个原理，即荧光背景不会改变，而拉曼散射则会因激光波长的周期性调制而发生位移。利用这一物理特性，以及拉曼信号的多通道锁定检测的重要性，调制技术满足了有效荧光扣除方法的要求。实际上，一旦完成同步和校准程序，就不需要用户进行干预，使得该方法成为在线的，并且比其他荧光抑制方法更耗时。我们分析了悬浮在荧光染料溶液中的 2 µm 大小聚苯乙烯珠的调制拉曼信号和位移激发拉曼差谱（SERDS）信号随调制率的变化。我们表明，在最高调制率下，调制拉曼光谱的信噪比比 SERDS 高 3 倍。为了最终评估调制拉曼光谱的实际优势，我们将该技术应用于中国仓鼠卵巢细胞（CHO）。具体来说，通过分析 CHO 细胞膜、细胞质和细胞核的单独光谱，我们证明了该方法能够从细胞中获得局部敏感的化学信息，远离干扰的荧光背景。特别是，拉曼数据的统计分析和使用 PCA（主成分分析）的分类表明，我们的方法允许我们以更高的灵敏度和特异性区分不同的细胞位置，避免使用标准背景程序获得的数据可能出现的错误解释。
