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

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.

The fluorescent imaging of plants is often hampered by background autofluorescence from chloroplasts, waxes and other metabolites, especially in the red part of the spectrum where chlorophyll dominates. This has greatly hindered the application of new super-resolution techniques to resolve the molecular make-up of sub-cellular systems in plant cells which is increasingly important for the understanding of fundamental processes of plant pathology. We will overcome this limitation by adapting a new super-resolution approach called DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) for the imaging of plant cells in a proof-of-concept study. Other super-resolution techniques (such as PALM/STORM) place severe constraints on dye choices due to photo-switching requirements. DNA-PAINT relaxes this and one can freely choose a dye whose emission has minimal overlap with plant autofluorescence but provides extremely high photon yields. Using imaging conditions so that 5-10K photons can be collected in individual localisation events using dyes emitting in the blue-green we will generate contrast for high-quality super-resolution imaging. We will also validate fully quantitative super-resolution imaging to count molecules in a mode called qPAINT and introduce a new method to perform the required in-situ calibrations using a novel label-free single molecule sensor that has been developed by one of us. To facilitate routine quantitative super-resolution imaging with Arabidopsis we will integrate the plant and calibration sensor into a microfluidic package (plant-in-a-chip) that establishes calibrated DNA-PAINT. This allows live cell imaging prior to in-chip fixation and staining and enables fully correlative live-cell diffraction limited plant imaging followed by fixed-cell super-resolution imaging with molecular resolution. Our next generation bioimaging tools will help resolve the molecular basis of pathogen invasion and plant cell defence systems.

Improved bioimaging tools for plant cell biology will have a broad relevance for a number of important stakeholders:

1. Crop Protection and Global Food Security

The outcomes of this research will have impact in the long-term for both crop protection and enhancement by providing bioimaging tools to obtain an improved understanding of pathogenic as well as beneficial symbiotic relationships between plants and pathogens. Strains of pathogenic microbes from many species currently challenge the genetic defences of high-yield crops established during the Green Revolution. The molecular basis of these adaptations is not currently understood. The bioimaging tools that we are developing here will make a major contribution to advance our ability to observe the molecular relationships underlying these fundamental interactions. We have identified the technological need and formulated our biological hypotheses through our work to understand pathogen-targeted secretion in plants (BB/M024172/1). The capability to resolve alterations in receptor configurations, effector-target interactions and sites of host-microbe interfaces will be of high value across the phytopathology field. This ability to understand how pathogens evade crop immune systems arms society against current and future threats to food security.

2. The Agrochemical Industry

While our current research tackles the fundamental nature of plant defence systems, findings obtained using our novel bioimaging tools are anticipated to provide results that will be of high value to the UK biotechnology sector in the long term. Agrochemical companies such as Syngenta support research into the molecular basis of pathogen virulence in order to identify new potential targets for the development of crop protection products. The BBSRC/Syngenta IPA award BB/M022900/1 (for which MD is a co-PI) is an example of an industrial partnership to isolate key virulence factors in the wheat pathogen Zymoseptoria tritici. Concurrent partnerships with Syngenta and the University of Exeter Physics department are exploring methods to directly image the distribution of fungicides within plant tissues. Our imaging technology outputs from this proposal will be employed to advance these studies and explore new options for enhancing crop defence.

3. Capacity Building

By providing excellent training and support to the PDRA employed to undertake this project we will be helping to secure the science base of the future. Outstanding researchers will be key to overcoming future tool challenges and the food security challenges that we are already facing at this time (and which are projected to increase massively over the next 50 years). The PDRA will complete the project with a unique blend of research skills including quantitative super-resolution imaging and new biosensor devices as well as DNA nanotechnology and microfluidics. This is a highly valuable skills package that will benefit future BBSRC funded research. The research fellow will acquire and refine a set of skills that should make them sought after for employment in biotechnology, pharmaceutical industry, advanced application development and a variety of research and development roles.

The types of benefit outlined above make a contribution to the nation's health and wealth and help to enhance quality of life and long term health.