Broadband coherent raman cell imaging for in situ protein co-localisation

光学显微镜的使用对于研究蛋白质在生物细胞中的相互作用和控制细胞功能的方式特别有吸引力，因为它们允许在细胞的实际环境中研究这些生物过程。用荧光分子标记蛋白质的能力使显微镜能够确定细胞中不同蛋白质的位置。这些蛋白质的空间“共定位”可以提供关于它们如何相互作用的有价值的信息。虽然这种方法非常成功，但荧光的使用确实存在一些固有的问题。我们的目标是研究另一种光谱成像技术，相干反斯托克斯拉曼散射（CARS）成像，作为荧光显微镜的替代技术，它提供了克服这些问题的潜力。CARS是一种非线性光学技术，可以测量与荧光强度相似的强振动信号。特别是，我们将实现宽带CARS，其中可以在每个扫描点测量CARS光谱，因为感兴趣的样品通过紧密聚焦的激光光斑扫描。我们将考虑使用传统的小荧光分子作为CARS标签，每个分子都有明确的CARS光谱。我们还将应用数值算法（主成分分析）将CARS光谱分离为不同标签的成分光谱。这将允许在每个扫描点同时测定多个标签的局部浓度，用于多重蛋白质共定位测定。我们将对固定细胞和组织切片进行初步测量，以评估该技术的能力，以便与我们的生物学合作者进一步应用，并进一步发展该技术，朝着体内成像的方向发展。

The use of light microscopy is particularly attractive for the study of the ways in which proteins interact in biological cells, and control cell function, since they permit the study of such biological processes within the actual environment of the cell. The ability to tag proteins with fluorescent molecules enables the determination, with microscopy, of the locations of different proteins in the cell. The spatial 'co-localisation' of these proteins can then provide valuable information about how they interact. Although this approach is enormously successful, the use of fluorescence does present a number of inherent problems. We aim to investigate another spectroscopic imaging technique, coherent anti-Stokes Raman scattering (CARS) imaging, as an alternative to fluorescence microscopy which offers the potential to overcome these problems. CARS is a non-linear optical technique which allows the measurement of strong vibrational signals, of similar strength to fluorescence. In particular, we will implement Broadband-CARS, in which a CARS spectrum can be measured at each scan point, as the sample of interest is scanned through a tightly focussed laser spot. We will consider the use of conventional small fluorescent molecules as CARS labels, each with a well defined CARS spectrum. We will also apply numerical algorithms (principle components analysis) to separate CARS spectra into the constituent spectra from different labels. This will allow the simultaneous determination at each scan point of the local concentration of a number of labels, for multiplexed protein co-localisation assays. We will perform preliminary measurements on fixed cells and tissue sections, to assess the capabilities of the technique for further application with our biological collaborators and for further development of the technique, towards in vivo imaging.