High-resolution imaging of the electric surface potential of biomolecular structures

自从17世纪Anton van Leeuwenhoek首次使用手工制作的光学显微镜观察生物细胞以来，显微镜技术一直是生物学中的重要工具。从那时起，显微镜学有了长足的发展，从简单的光学透镜显微镜发展到使用除光以外的物理机制的显微镜，如电子束或机械扫描探针。在扫描探针显微镜中，一个比生物细胞小得多的微小的微加工尖端被扫描到物体的表面，同时紧密地跟随物体的表面轮廓。然后，计算机分析提供物体表面的三维图像，物体可以是细胞，也可以是蛋白质或DNA分子。与电子显微镜不同，扫描探针显微镜在空气和水中（大多数活细胞的自然环境）都有很大的优势，而且样品不需要涂上金属。近年来，材料和半导体科学家开发了扫描探针显微镜的一种变体，称为开尔文探针力显微镜。这种新方法不仅可以成像表面的粗糙度和结构，还可以成像它们的电学性质，这为材料的组成和带电分子的高分辨率位置提供了重要的额外线索。该方法在检测前不需要对样品进行任何化学或物理改性，而且非常敏感。这个技术驱动的研究项目位于生物和物理科学的交汇处，旨在将开尔文探针力显微镜应用于生物学。到目前为止，开尔文探针力显微镜只能在空气或真空中工作，而大多数生物样品需要在浸入水中时进行研究。我们的目标是开发新的仪器，使我们能够在水中进行高分辨率的开尔文探针力显微镜测量。这将包括设计和制造新的显微镜尖端，以及对市售仪器的技术修改。为了评估开尔文探针力显微镜是否可以在水中进行高分辨率操作和成像，我们将创建具有定义和规则几何形状的二维电荷模式。这些模型结构将使用自然产生的蛋白质获得，这些蛋白质具有自组装成具有重复特征的大晶体片的能力。我们将通过蛋白质的基因工程将常规电荷引入这些蛋白质薄片。我们相信，开尔文探针力显微镜在水中测量的成功扩展将为表面电荷起重要作用的生物学研究开辟新的途径。例如离子通道的可视化，嵌入细胞膜的带电分子或活细胞中的整个细胞膜域。这种仪器将对生物学家、生物医学科学家和生物物理学家有很大的好处，他们将能够获得生理条件下静电表面电位的空间图像，并可能导致扫描探针仪器制造商将新的研究仪器商业化。

Microscopical techniques are important tools in biology ever since the 17th century, when Anton van Leeuwenhoek observed biological cells for the first time using a handcrafted, optical microscope. Since then microscopy has evolved substantially, from simple optical lens microscopes to microscopes that use physical mechanisms other than light, such as electron beams or mechanical scanning-probes. In scanning-probe microscopy a tiny, microfabricated tip, which is much smaller than a biological cell, is scanned over a surface of an object while closely following its surface contours. Computer analysis then provides a three-dimensional image of the surface of the object, which can be a cell, but also protein or DNA molecules. Unlike electron microscopy, scanning-probe microscopy has the great advantage to work in air as well as in water, the natural environment of most living cells, and the sample does not need to be coated with a metal. In recent years, a variant of scanning-probe microscopy, termed Kelvin-probe Force Microscopy has been developed by materials and semiconductor scientists. This novel method can image not only the roughness and structure of surfaces but also their electrical properties, which provides important, additional clues about the composition of materials and the location of charged molecules at high resolution. The method does not need any chemical or physical modification of the sample prior to investigation and it is extremely sensitive. This technology-driven research project, which is located at the interface of biological and physical sciences, aims to adapt Kelvin-probe Force Microscopy for use in biology. So far, Kelvin-probe Force Microscopy works only in air or vacuum whereas most biological samples need to be investigated when immersed in water. Our objective is to develop new instrumentation to enable us to perform Kelvin-probe Force Microscopy measurements at high resolution in water. This will encompass the design and fabrication of new microscope tips as well as technical modifications of commercially available instruments. To evaluate whether Kelvin-probe Force Microscopy can operate and image at high resolution in water, we will create two-dimensional patterns of electrical charges with defined and regular geometry. These model structures will be obtained using naturally occurring proteins which have the ability to self-assemble into large crystalline sheets with repeating features. We will introduce regular charges into these protein sheets via genetic-engineering of the protein. We believe that the successful expansion of Kelvin-probe Force Microscopy to measurements in water will open new routes for research in biology, where surface charges play an important role. Examples are the visualisation of ion-channels, charged molecules embedded in cell membranes or entire cell membrane domains in living cells. This instrumentation will be of great benefit to biologists, biomedical scientists and biophysicists who will be able to obtain a spatial image of the electrostatic surface potential under physiological conditions, and could possibly lead to commercialisation of new research instruments by scanning-probe instrument manufacturers.