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相互作用的方式和控制细胞功能特别有吸引力，因为它们允许在细胞的实际环境中研究这种生物过程。用荧光分子标记蛋白质的能力使人们能够用显微镜确定不同蛋白质在细胞中的位置。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。尽管这种方法非常成功，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方案，它提供了克服这些问题的可能性。CARS是一种非线性光学技术，可以测量强度类似于荧光的强振动信号。特别是，我们将实施宽带-CARS，在每个扫描点都可以测量CARS光谱，因为感兴趣的样品通过紧密聚焦的激光光斑进行扫描。我们将考虑使用传统的小荧光分子作为汽车标签，每个标签都有明确的CARS光谱。我们还将应用数值算法(主成分分析)将CARS光谱分离为来自不同标签的组成光谱。这将允许在每个扫描点同时确定多个标记的局部浓度，用于多重蛋白质共定位分析。我们将对固定的细胞和组织切片进行初步测量，以评估该技术的能力，以便与我们的生物合作者进一步应用，并进一步开发该技术，走向活体成像。

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.