Phyto-optofluidics - A quantitative super-resolution imaging approach for next generation plant physiology research

荧光显微镜被广泛用作研究细胞和组织的生物学和生物物理特性的敏感工具，因为它提供了出色的对比度，高分辨率，并且可以对在细胞和组织中起重要作用的各种类型的生物分子特别敏感。直到大约10年前，人们还认为荧光成像与其他类型的光学显微镜一样，具有固有的局限性，因为光是一种电磁波，因此其分辨率仅限于光波长的一半，即约250 nm。因此，光学显微镜无法直接分辨通常只有几纳米大小的生物分子。这种限制已经被称为“超分辨率”技术的新型显微镜所克服。这些超分辨率技术为观察复杂的生物系统（如细胞和组织）打开了一扇窗户，因为它们提供了对这些系统分子结构的直接观察。这一知识在植物生物学中变得尤为重要，因为我们正试图了解植物受到病原体影响时发生的过程。植物对这些挑战的反应取决于特定类型的生物分子的作用，我们需要一种方法来检测这些分子的小群体的协同作用如何参与重要的植物防御机制。这些信息对于开发保护植物的新方法至关重要，也是当前粮食安全研究工作的一个关键方面。尽管使用这些新的高分辨率显微镜技术观察植物中的分子很重要，但植物在显微镜下照射时产生的强烈背景信号阻碍了这一点。捕获绿色光的叶绿素是一个特别的问题，最近有人提出，我们对DNA及其形成双链体（通常形成众所周知的双螺旋）的特性的详细了解，可以用来调整发射彩色光的分子（即染料分子）与生物学家用来附着在特定生物分子上的标记分子之间的分子相互作用。在这个项目中，我们将使用这种方法使单个分子特别明亮，以便它们可以在叶绿素产生的植物细胞背景下被看到。由于新的合成DNA方法给我们带来的灵活性，我们可以使用背景信号较弱的颜色范围。在这个项目中，我们将首次展示新的基于DNA的方法如何克服以前在植物样品中成像的问题，并显示植物细胞中对抵抗感染至关重要的分子。我们将开发的新成像工具的另一个方面是成像的定量模式，以便可以直接计数分子的数量，这对于细胞生物学的数学理解至关重要。在我们的新方法中，分子计数模式将通过采用一种新型传感器来简化，该传感器提供了一种计数标准，我们可以将其用于任何定量方法中的重要步骤，即校准。通过将校准传感器与我们的DNA成像技术相结合，常规校准变得相对简单，有助于实现分子数量的常规和准确测量。最后，我们将安排我们新成像技术的组件，以便植物可以在一个小型实验室中生长，允许实验人员将营养液流过我们的植物，以支持正常的植物生长和发育。实验人员可以在对植物细胞成像的同时引入病原体分子。结合新的成像工具，这将使植物在不断变化的环境中的现实和良好的控制研究成为地球上植物生命的微型模型。

Fluorescence microscopy is widely used as a sensitive tool to investigate the biology and biophysical properties of cells and tissues since it provides exceptional contrast, high resolution and can be made specifically sensitive to individual types of biomolecules that play vital roles in cells and tissues. Until about 10 years ago it was thought that fluorescence imaging, like other types of optical microscopy, is inherently limited because light is a type of electromagnetic wave and its resolution is therefore limited to about half the wavelength of light, or ~250 nm. Light microscopy would therefore be incapable of directly resolving biomolecules that are typically only a few nanometres in size. This limitation has been overcome by new types of microscopy that are called "super-resolution" techniques.These super-resolution techniques have opened a window into complex biological systems such as cells and tissues because they provide a direct view of the molecular structure of these systems. This knowledge is becoming especially important in plant biology as we are attempting to understand the processes that occur when a plant is affected by a pathogen. The response of the plant to such challenges depends on the action of particular types of biomolecules and we need a way to detect how the concerted action of small groups of such molecules are involved in vital plant defence mechanisms. This information is critical to developing new ways to protect plants and a key aspect of current food security research efforts. Despite the importance of seeing molecules in plants using these new high-resolution microscopy techniques this has been hampered by the strong background signals that plants generate when they are illuminated under the microscope. Green light-harvesting chlorophyll is a particular problem.Recently it has been suggested that our detailed knowledge of DNA and its properties in forming duplexes (that typically form the well-known double-helix) can be used to tailor the molecular interactions between molecules that emit coloured light, i.e. dye molecules, and the marker molecules that biologists use to attach to specific biomolecules. In this project we will use this approach to make individual molecules especially bright so that they can be seen against the plant cell backgrounds arising from chlorophyll. Due to the flexibility that the new synthetic DNA approach gives us we can use a colour range where the background signals are weaker.In this project we will for the first time show how the new DNA based approach can overcome previous problems with imaging in plant samples and show molecules in plant cells that are critical for resisting infections. An additional aspect of the new imaging tools that we will develop is a quantitative mode of imaging so that the number of molecules can be directly counted which is critical for mathematical understanding in cell biology. The molecular counting mode will be simplified in our new approach by employing a new type of sensor that provides a counting standard that we can use for an important step in any quantitative method, namely calibration. By integrating the calibration sensor with our DNA imaging technique routine calibration becomes a comparatively straightforward task which helps achieve routine and accurate measurements of molecule numbers.Finally, we will arrange the components of our new imaging technique so that the plants can be grown in a small experimental chamber that allows experimenters to flow nutrients past our plants to support normal plant growth and development. Experimenters can introduce molecules from pathogens while imaging the plant cells. In combination with the new imaging tools this will enable realistic and well-controlled studies of plants in changing environments as a miniature model of plant life on earth.