Live-cell, deep-tissue, low-light, 3D-STED confocal microscopy: a super-resolution imaging platform specifically designed for plant science.

Microscopy is one of the oldest methods used to study complex biological processes and has seen major developments since the creation of the first microscopes in the late 16th century. Fluorescence microscopy has allowed many breakthrough discoveries by giving scientists the ability to locate and track specific molecules within cells. All light microscopes are limited by the physical law of diffraction. However, the development of super resolution (SR)microscopy, which was recognized by a Nobel prize to E.Betzig, S. Hell and E. Moerner in 2014, has revolutionised the range of imaging that can be achieved and biological questions that can be answered. After two decades of combined improvements in these technologies and in associated computing power, SR microscopes are becoming more accessible and can now be customised for individual biological systems.

The vast majority of the current super resolution systems have been designed and calibrated by their manufacturers for studies using human and animal cells. However, plant cells are larger and more complex than animal cells, contain thick cell walls and emit significant auto-fluorescence across the visible spectrum. This makes SR imaging extremely challenging in live plant cells and, consequently, few manufacturers have designed microscopes suitable for this purpose. In turn, this lack of SR imaging of plant cells in the UK is impeding breakthrough discoveries and ultimately restricting plant science. The James Hutton Institute has world-renowned expertise in plant and agricultural research. One area of research is to develop crops that are more resilient to climate change and resistant to pests and diseases; subjects that require an understanding of cellular processes to help mitigate risks to plant health and agriculture. Our existing imaging facility has been a core component in our cell and molecular research but advances in microscope technology mean that we need access to super-resolution microscopy techniques to remain at the forefront of cell biology research.

We propose to purchase a super-resolution microscope, providing the science community in Scotland and the UK with a powerful system optimised for use in plants. We have found that the STED technology provided by the Abberior Facility-Line 3D-STED microscope was by far the best equipment in recent tests, allowing imaging of both fixed and live plant samples and providing up to 50nm resolution. At present there are no similar microscopes in the UK that are optimised for plant cell imaging, and legislation and plant health regulations restrict us from taking many of our samples off-site to utilise other facilities. Since we have excellent (and licensed) plant growth facilities at JHI it makes sense to install a SR microscope in our existing facility and make it available to the wider biological research community across Tayside and the UK. In time the equipment will be moved to the Advanced Plant Growth Centre; a purpose-built, world-class plant phenotyping facility which is currently being built on the JHI site. Both Dundee and St Andrews Universities are partners in this bid and many researchers (from a wide range of disciplines) have submitted requests to use this resource if it becomes available, covering projects in the areas of sustainable agriculture and food, understanding the rules of life, an integrated understanding of health and transformative technologies. Researchers who receive training to use these techniques and equipment will benefit from continuing professional development, the facility and its core staff will benefit from exposure to new technology and expansion and diversification of our user group, the BBSRC will benefit from enhanced research outputs and end-users will benefit from the outcomes of the research.

The UK currently lacks a super-resolution (SR) microscope facility aimed primarily at the plant science community. To address the increasing demand, we propose to purchase and host a SR microscope, optimised for use with plant samples but suitable for all biologists. Several SR techniques and microscope systems are available. Structured Illumination Microscopy (SIM) can achieve 100nm resolution in XY but only 250nm in Z so is not ideal for large samples. Stochastic optical reconstruction microscopy (STORM) resolution is better, providing single molecule resolution of ~20 nm, but imaging biological samples is very challenging where autofluorescence is present. Stimulated emission depletion (STED) technology can achieve a lateral resolution of 50-100nm by exploiting the non-linear response of fluorophores.

JHI researchers and collaborators have tested several techniques and found STED to be the most suitable for plants. In our preliminary work, we have found that the 3D-STED and Expert Devices technology of the Abberior Facility-Line allowed imaging of fixed and live plant samples and successfully separated chlorophyll autofluorescence from fluorescent protein signals; a prerequisite for plant work. In tests the system obtained high quality images in both confocal and STED modes, even for fast-moving structures in live cells. We will purchase the first 3D-STED microscope in the UK constructed for plant imaging that works beautifully on both fixed samples and live cells in intact tissues. While specialised for plant samples, the system will be suitable for nanoscale studies of other organisms to help meet the increasing demand for SR imaging in the UK bioscience community. The microscope has been selected to be user-friendly, with facile software; well-suited to a diverse, multi-user facility. The modular design allows upgrades and expansion. Outputs will push imaging boundaries, drive scientific discoveries, engage researchers and enable education.