van Manen Henk-Jan, Otto Cees
AkzoNobel Research, Development & Innovation Measurement & Analytical Science Molecular Spectroscopy Group, Zutphenseweg 10, 7418 AJ, Deventer, The Netherlands.
Methods Mol Biol. 2011;680:45-60. doi: 10.1007/978-1-60761-901-7_3.
Raman spectral imaging is a label-free, noninvasive optical technique to visualize the spatial distribution of biomolecules such as DNA, RNA, proteins, and lipids in cells and tissues. Although Raman imaging has been successfully used in the last 5 years on single cells, an important drawback of this technique is that it is traditionally regarded as incompatible with fluorescence microscopy. This is because fluorescence signals from fluorophore-labeled cells usually overwhelm the orders of magnitude weaker Raman signals from cellular biomolecules. However, we have recently shown that both nonresonance and resonance Raman microscopy can be combined with fluorescence microscopy on the same cells by spectrally separating fluorescence emission from Raman scattering. The fluorophores that are used in this case are semiconductor quantum dots (QDs), which have suitable properties in hybrid Raman-fluorescence experiments, in particular a large separation between absorption and emission wavelengths. We envisage that the combination of fluorescence microscopy with Raman spectroscopy or imaging on cells will lead to new application in cell biology. Here, we will describe detailed protocols for performing hybrid Raman-fluorescence experiments on single QD-labeled cells.
拉曼光谱成像技术是一种无需标记、非侵入性的光学技术，用于可视化细胞和组织中生物分子（如DNA、RNA、蛋白质和脂质）的空间分布。尽管拉曼成像技术在过去五年中已成功应用于单细胞研究，但该技术的一个重要缺点是，传统上认为它与荧光显微镜不兼容。这是因为来自荧光团标记细胞的荧光信号通常会淹没来自细胞生物分子的强度弱几个数量级的拉曼信号。然而，我们最近表明，通过在光谱上分离荧光发射和拉曼散射，非共振和共振拉曼显微镜都可以与同一细胞上的荧光显微镜相结合。在这种情况下使用的荧光团是半导体量子点（QD），它在拉曼-荧光混合实验中具有合适的特性，特别是吸收和发射波长之间有很大的间隔。我们设想，荧光显微镜与细胞上的拉曼光谱或成像技术相结合将在细胞生物学中带来新的应用。在这里，我们将描述在单个量子点标记细胞上进行拉曼-荧光混合实验的详细方案。
