High-resolution low-voltage TEM for imaging the process of mineralization at the TMV/inorganic interface: Towards understanding the mechanical properties of bio/inorganic multilayer systems

生物矿化是组织构建和硬化的自然过程，其物理和化学性质优于几种合成产物。值得注意的是，有人试图通过生物激发矿化合成类似技术相关的混合材料。在现有的项目中，我们希望通过高分辨率的有机/无机胶线成像，更好地了解硫化锌（ZnS）和氧化锌（ZnO）在生物模板烟草花叶病毒（TMV）上的蛋白矿化机制。所获得的见解将有助于控制该边界表面的结构，这对于合成具有定制性能的纳米功能材料至关重要。对于常用的有机材料成像技术，通过对样品进行负染色，其分辨率在几纳米范围内，或者在300 kV的加速电压下将其嵌入玻璃化冰中，分辨率高达0.4 nm，我们依靠2016年春季新开发的低压透射电子显微镜（TEM）来对表面边界进行成像。由于物镜的球面校正和色差校正，在20- 80kv的加速电压下可以获得高分辨率的胶线图像。在这个项目的框架内，将研究精细的样品制备方法，如石墨烯三明治和/或氘化样品，以成像有机/无机边界表面。此外，该项目的另一个目标是原位揭示生物激发矿化是否始于非均质成核或均质成核。在前期工作中，Stuttgart小组完成了在烟草花叶病毒（TMV）上选择性沉积ZnS和ZnO的矿化过程。在本项目框架内，将分别以TMV/ZnS和TMV/ZnO为基础，制备和研究多层珠状结构（MLS）。对野生型（wt） TMV （wt-TMV/ZnO）和TMV突变体TMV_Cys （TMV_Cys/ZnO）以及在边界表面添加硫醇的ZnO基MLS的力学性能研究表明，含有突变体的MLS的硬度比wt基MLS高。这些MLS也将被我们的低压透射电镜成像，以分析蛋白质-矿物边界表面的超微结构差异，并将其与机械性能联系起来。为了深入了解病毒表面和内部通道的矿化情况，另外还计划组装小病毒样颗粒（VLP）并将其矿化成垂直定向阵列。

Biomineralization is the natural process of construction and induration of tissues, which outmatch several synthesized products in their physical and chemical properties. Appreciably, one tries to synthesize comparable technical relevant hybrid materials by bio-inspired mineralization. In the existing project we would like to contribute to a better understanding of the mechanism of protein based mineralization of zinc sulfide (ZnS) and zinc oxide (ZnO) on the biotemplate tobacco mosaic virus (TMV) by imaging the organic/inorganic glue line with high-resolution. The obtained insights will help to control the structure of this boundary surface, which are essential for synthesis of nano-sized functional material with tailored properties.Toward the commonly used techniques to image organic materials by negative staining of the specimen, which leads to resolutions in the range of several nanometer or embedding them in vitrified ice with a resolution up to 0.4 nm at acceleration voltages of 300 kV, we count on our newly developed low-voltage transmission electron microscope (TEM) available in spring 2016 to image the surface boundary. High resolution images of the glue line at acceleration voltages of 20-80 kV is possible due to not only spherical but also chromatic aberration correction of the objective lens. In the frame of this project elaborate sample preparation methods like graphene-sandwiching and/or deuterating the specimen will be investigated to image the organic/inorganic boundary surface. Additionally, a further goal of this project is to reveal in situ if the bio-inspired mineralization begins with heterogeneous or homogenous nucleation.In preliminary work of the Stuttgart group, mineralization procedures for selective deposition of ZnS and ZnO on tobacco mosaic virus (TMV) were accomplished. In the frame of this project multilayered nacre-like structures (MLS) on the base of TMV/ZnS and TMV/ZnO, respectively will be produced and investigated. The mechanical properties investigations of ZnO-based MLS with wildtype (wt) TMV (wt-TMV/ZnO) and the TMV mutant TMV_Cys (TMV_Cys/ZnO) and therefore with additional thiols in the boundary surface revealed that the MLS containing the mutant showed an increase in hardness compared to wt-based one. Those MLS will also be imaged with our low-voltage TEM to analyze ultrastructural disparity of the protein-mineral-boundary surface and to correlate that with mechanical properties. To gain an insight with the TEM into the mineralization at the surface as well as in the inner channel of the virus it is additionally planned to assemble small virus-like particles (VLP) and mineralized them in vertical oriented arrays.