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

自从17世纪Anton van Leeuwenhoek第一次使用手工制作的光学显微镜观察生物细胞以来，显微技术一直是生物学中的重要工具。从那时起，显微镜有了很大的发展，从简单的光学透镜显微镜到使用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、微型制造的尖端被扫描到物体的表面上，同时紧紧跟随其表面轮廓。然后，计算机分析会提供物体表面的三维图像，可以是细胞，也可以是蛋白质或DNA分子。与电子显微镜不同，扫描探针显微镜具有在空气和水中工作的巨大优势，而水是大多数活细胞的自然环境，而且样品不需要覆盖金属。近年来，材料和半导体科学家发展了一种称为开尔文探针力显微镜的扫描探针显微镜。这种新的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，这为高分辨率下材料的组成和带电分子的位置提供了重要的额外线索。该方法在研究前不需要对样品进行任何化学或物理修饰，且具有极高的灵敏度。这项由技术驱动的研究项目位于生物和物理科学的交界处，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，而大多数生物样品在浸入水中时需要进行研究。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，以及对商业仪器进行技术改造。为了评估开尔文探针力显微镜能否在水中以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电荷的二维图案。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片的能力。我们将通过对蛋白质进行基因工程，将有规律的电荷引入这些蛋白质片层。我们相信，开尔文-探针力显微镜的成功扩展到水中的测量将为生物学研究开辟新的途径，表面电荷在生物学研究中发挥着重要作用。例如，离子通道的可视化，带电分子嵌入细胞膜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大有裨益，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.