Lieber Chad A, Mahadevan-Jansen Anita
Department of Biomedical Engineering, Vanderbilt University, Station B, Box 351631, Nashville, Tennessee 37235, USA.
Appl Spectrosc. 2003 Nov;57(11):1363-7. doi: 10.1366/000370203322554518.
One of the challenges of using Raman spectroscopy for biological applications is the inherent fluorescence generated by many biological molecules that underlies the measured spectra. This fluorescence can sometimes be several orders of magnitude more intense than the weak Raman scatter, and its presence must be minimized in order to resolve and analyze the Raman spectrum. Several techniques involving hardware and software have been devised for this purpose; these include the use of wavelength shifting, time gating, frequency-domain filtering, first- and second-order derivatives, and simple curve fitting of the broadband variation with a high-order polynomial. Of these, polynomial fitting has been found to be a simple but effective method. However, this technique typically requires user intervention and thus is time consuming and prone to variability. An automated method for fluorescence subtraction, based on a modification to least-squares polynomial curve fitting, is described. Results indicate that the presented automated method is proficient in fluorescence subtraction, repeatability, and in retention of Raman spectral lineshapes.
将拉曼光谱用于生物应用面临的挑战之一是，许多生物分子产生的固有荧光构成了测量光谱的基础。这种荧光强度有时可能比微弱的拉曼散射强几个数量级，为了解析和分析拉曼光谱，必须尽量减少其影响。为此已设计出多种涉及硬件和软件的技术；这些技术包括使用波长移位、时间选通、频域滤波、一阶和二阶导数，以及用高阶多项式对宽带变化进行简单曲线拟合。其中，多项式拟合已被证明是一种简单而有效的方法。然而，该技术通常需要用户干预，因此既耗时又容易出现变化。本文描述了一种基于对最小二乘多项式曲线拟合进行改进的荧光扣除自动化方法。结果表明，所提出的自动化方法在荧光扣除、重复性以及保留拉曼光谱线形方面表现出色。
