Generation of luminescent materials through living micoalgae

在这个后续项目中，我们希望进一步研究海洋藻草草的生物矿化过程。我们想要揭示稀土元素Tb从周围的介质进入球石产生的细胞内球囊(Cv)的途径。本项目的第一部分证实了Tb3+在球石中的掺入，这些结果表明，在细胞内的cv中，Tb3+被掺入到正在生长的方解石球石中。研究Tb3+通过细胞的途径的优势在于它在光激发下的特殊荧光。为了研究Tb3+在细胞内的转运和途径，我们想要使用两种不同的方法：(A)我们想要用2D电泳法来鉴定参与Tb通过细胞转运的蛋白质。在此，我们想要比较钙化的湖北E.huxleyi菌株CCMP1516和非钙化的菌株CCMP1516NC在不同Tb3+和/或Ca~(2+)含量下的蛋白质组。(B)，我们希望通过共聚焦显微镜和电子显微镜技术来揭示结核病在从细胞膜到脑血管的过程中积累在细胞的哪些细胞器和/或隔室。首先，我们用共聚焦显微镜观察在培养液中加入Tb3+后的活细胞。我们希望使用滤光片跟踪Tb3+在细胞中的分布，从而可以专门可视化Tb3+的发射。由于细胞内球石的形成发生在1小时到2小时内，我们将评估在这段时间内Tb3+在细胞内积累的位置。当我们观察到其在细胞内的特定部位积聚时，将相应的细胞取样并在低温条件下进一步处理以进行透射电子显微镜的研究，即首先用高压冷冻机(HPF)冷冻细胞，冷冻替代细胞，切割后用Cryo TEM/EDX和配备CL的透射电子显微镜进行分析。

In this follow-up project we want to further investigate biomineralization processes in the marine alga Emiliania huxleyi. We want to reveal the pathway of the lanthanide terbium from the surrounding medium into the intracellular coccolith vesicle (cv), where the coccolith is produced. The incorporation of Tb3+ into the coccoliths was proven in part 1 of the project, these results showed, that Tb3+ is incorporated into the growing calcite coccolith in the cv inside the cells. The advantage of investigating the pathway of Tb3+ through the cells is its specific fluorescence when excited with light. For the investigation of the transport and pathway of Tb3+ in the cell, we want to use two different approaches: (A), we want to identify the proteins which are involved in the transportation of Tb through the cell using 2D electrophoresis. Here we want to compare the proteomes of the calcifying E. huxleyi strain CCMP1516 with the non-calcifying strain CCMP1516NC and at different amounts of Tb3+ and/or Ca2+ in the medium. (B), we want to reveal in which organelles and/or compartments of the cells the Tb is accumulated during its way from the cell membrane to the cv by Confocal microscopy and Electron microscopy techniques. First, we will observe the living cells after adding Tb3+ to the medium using a confocal microscope. We want to track the distribution of the Tb3+ in the cells using a filter, thereby we can visualize exclusively the Tb3+ emission. Since the formation of the intracellular coccoliths occurs within 1h to 2h, we will evaluate, where inside the cells the Tb3+ is accumulated during this time period. When we can observe its accumulation at a specific site in the cells, the corresponding cells will be sampled and further treated for the investigation by TEM under cryo-conditions, i.e. first freezing the cells with high pressure freezer (HPF), cryo-substitute them and after cutting analyze them using Cryo TEM/EDX and a TEM equipped with CL.