In vivo- and in vitro-analysis of single diatom cells/ cell walls by optical imaging using Raman spectroscopy and non-linear microscopy

拉曼光谱是一种重要的分析工具，可以应用于从整数单元非破坏性地获得分子信息。基于我们正在进行的关于单硅藻细胞壁研究的合作，拉曼光谱方法将在拟议的项目中开发和使用，以研究细胞壁组成以及在形成微米和纳米结构硅藻细胞壁期间发生的代谢过程。这些测量包括对单个提取的细胞壁的研究以及通过显微操作系统与成像技术相结合对活细胞的研究。基于二次谐波产生，双光子荧光和相干反斯托克斯拉曼散射的非线性显微镜将被额外应用。该项目的目的是研究在细胞周期中发现的有机分子的性质和组成的变化。此外，将监测细胞对生长培养基组成的确定变化的反应。光阱允许在活的单个硅藻细胞上进行拉曼实验。将通过在微流体环境中结合光阱和拉曼光谱进行体内在线监测。此外，还将结合其他分析技术（ICP-OES、NMR）对提取的单细胞壁进行拉曼显微镜检查，特别是针对细胞壁中发现的无机组分。化学计量学对数据的解释很重要。

Raman spectroscopy is an important analytical tool which can be applied in order to non-destructively obtain molecular information from integer cells. Based on our ongoing collaboration concerning the study of single diatom cell walls, Raman spectroscopic methods will be developed and used within the proposed project in order to study the cell wall composition as well as the metabolic processes taking place during the formation of the micro- and nanostructured diatom cell walls. The measurements include the study of single, extracted cell walls as well as the study of living cells by the combination of micromanipulation systems with imaging techniques. Non-linear microscopy based on second harmonic generation, two-photon fluorescence and coherent anti-Stokes Raman scattering will be applied additionally. The aim of the project is the investigation of variations of the nature and composition of organic molecules found during the cell cycle. Furthermore, the response of the cells upon defined variations of the composition of the growth medium will be monitored. Optical traps allow Raman experiments on living single diatom cells. In vivo online monitoring will be carried out by combining optical traps and Raman spectroscopy in a microfluidics environment. Furthermore, Raman microscopy will also be performed on extracted single cell walls in combination with other analytical techniques (ICP-OES, NMR) especially with respect to the inorganic components found in the cell walls. Chemometrics will be important for the interpretation of the data.