Combining coherent Raman scattering with fluorescence microscopy to investigate sugar uptake in living cells

糖是生命中必不可少的分子。它们是由特殊的膜转运蛋白在细胞膜上主动运输的，是糖生物合成的主要能源和碳来源。糖基化，作为一种普遍存在的翻译后修饰，早已为人所知。所有的细胞都覆盖着一层密集而复杂的聚糖层。这种所谓的糖萼参与了多种过程，包括信号传递、细胞黏附和基于多糖驱动的模式识别的免疫。尽管它很重要，但由于糖链结构的非凡变异性以及糖基化的动态性质，人们对糖基化的组成、结构和精确功能知之甚少。糖的摄取如何影响细胞糖基化的化学成分，目前还没有答案。组合结构方法已经确定了越来越多的单糖和多糖结构，但它们仅限于体外分析。代谢性低聚糖工程允许在体内对糖部分进行荧光成像。然而，糖的标签仍然具有挑战性。荧光标签会干扰糖的生物功能。在生理条件下，糖类物种的胞内浓度从NM到Mm不等。单分子成像不适合同时显示不同糖种的低浓度和高浓度，或同时显示所涉及的膜转运体和底物。尽管天然糖在生物医学上具有巨大的重要性，但我们缺乏一种高速、多路成像技术来研究天然糖在摄取、代谢和储存过程中作为原位多糖的作用。我们建议建立受激拉曼显微镜(SRS)，这是我在博士期间开创的，作为糖的化学图谱的工具。我将设计一个尖端的成像平台，将单分子活细胞成像与宽带受激拉曼显微镜相结合。它致力于体内多路成像，在高浓度下具有化学特异性，同时具有单分子灵敏度。我将建立已开发的方法来研究细胞内糖的吸收和储存。这一结合的方法用于定量细胞分辨率的过剩转运蛋白介导的糖摄取。此外，我们将研究MOE中插入的拉曼活性部分是否影响亲和力和摄取，以及非天然糖是主动吸收还是通过被动扩散。通过研究细胞中糖的储存，我们最终的目标是建立这种方法作为细胞糖萼的一种化学表征方法。

Sugars are essential molecules in life. Being actively transported over the cell membrane by specialized membrane transporters, they represent a major source of energy and carbon in glycan biosynthesis. Glycosylation, as ubiquitous post-translational modification has been known for a long time. All cells are covered with a dense and complex layer of glycans. This so-called glycocalyx is involved in various processes including signalling, cell-adhesion and immunity based on glycan-driven pattern recognition. Despite its importance, only little is known about the composition, structure and precise function of the glycocalyx due to the extraordinary variability of glycan structures as well as dynamic nature of glycosylation. It is unanswered, how sugar uptake influences the chemical composition of the cellular glycocalyx. Combined structural approaches have identified a growing catalogue of monosaccharides and glycan structures however they are restricted to ex vivo analysis. Metabolic oligosaccharide engineering allowed for fluorescence imaging of sugar moieties in vivo. Nevertheless, labelling of sugars remains challenging. Fluorescent labels can interfere with the sugar’s biological function. Sugars species are present at intracellular concentrations ranging from nM to mM range under physiological conditions. Single-molecule imaging is not suited to simultaneously visualize low and high concentrations of different sugar species, or the involved membrane transporter and substrate at the same time. Despite its immense biomedical importance, we lack a high-speed, multiplexed imaging technology that allows for studying natural sugars during uptake, metabolism and storage as glycans in situ. We propose to establish stimulated Raman microscopy (SRS), which I pioneered during my PhD, as tool for chemical mapping of sugars. I will design a cutting-edge imaging platform, that combines single-molecule live cell imaging with broadband stimulated Raman microscopy. It is dedicated for multiplexed in vivo imaging with chemical specificity at high concentrations and simultaneous single-molecule sensitivity. I will establish the developed methodology to investigate uptake and storage of sugar within the cell. The combined approach serves for the quantification of sugar uptake mediated by GLUT transporters with cellular resolution. Moreover, we will study whether inserted Raman-active moieties in MOE affect the affinity and uptake and if unnatural sugars are taken up actively or via passive diffusion. By investigating the cellular storage of sugars, we ultimately aim to establish this approach as a chemical characterization method for the cellular glycocalyx.