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

点击翻译按钮获取中文摘要

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.