Light Sheet

Food security represents a major global issue. Crop production has to double by 2050 to keep pace with a global population increasing to 9 billion. This target is even more challenging given the impact of climate change on water availability and the need to reduce fertilizer inputs to make agriculture become more environmentally sustainable. Research to meet these challenges is often based on studies using simpler plants which can reveal insights as to how important traits such as root growth and branching, water movement, seed germination and pollen release can be improved in crops. A new type of microscope (using a technique called Light Sheet Fluorescence Microscopy or LSFM) has recently been developed that, for the first time, allows the long-term study of plant growth and development and gene expression. These microscopes use a thin sheet of laser light to illuminate a specimen. Only the measured part of the sample is illuminated by the sheet, making the process less stressful and allowing imaging of living plants for several days.

The Zeiss Z.1 is the first commercially available LSFM. This imaging approach has not been widely used to study plants because the first machines lacked the ability to grow plants within the microscope. The microscope we have designed with the manufacturers will be fitted with temperature and light controls and will be one of the first light sheet microscopes in the world (and the first in the UK) to be specifically optimized for plant science.

We will use this unique tool to look at many biological questions of relevance to food security including:

1. Root responses to environmental signals. Roots adapt their growth in response to environmental signals like nutrients and water to optimise foraging in the soil for these important resources. LSFM images will allow for the first time visualization in 4-D of foraging processes such as hydrotropism when roots grow towards a water source.

2. Root vascular patterning for increased water use efficiency. The vascular tissues of the root represent the main system for moving water and nutrients. The LSFM images would provide important information into the activity of genes controlling vascular tissue identity, and help efforts to engineer lines with alternate vascular patterns that may have greater water use efficiency.

3. Seed germination occurs first by the seed coat rupturing, followed by the rupture of the endosperm (a coating of live cells). The process of endosperm stretching and rupture is difficult to study due to their sensitivity to humidity and temperature. This can be controlled in the LSFM, allowing tracking of cell geometry and markers over time.

4. Seeds also provide a major nutrient source for plants and animals. We would use the LSFM to understand how ovules develop within seeds, with the aim of maximising yield of oilseeds.

5. Anther and pollen development. The control of pollen viability and the release of functional pollen are critical for fertilization and crop yield. The LSFM will be used to visualize fluorescently labelled molecules with high resolution and with time, from pollen wall deposition, through anther opening, to pollen release.

As we move from understanding simple regulatory networks in simple systems to uncovering the complexity of non-linear regulatory networks in complex tissues, we need an increasing amount of information about the tissue geometry itself and about the spatio-temporal expression of key genes. Over the last few decades this information has primarily been gathered using confocal microscopy. However, this technique has many drawbacks that centre around, the two dimensional mounting of samples on glass slides, the phototoxicity required to perform long imaging and the slow rate of data acquisition meaning that samples suffer damage and photobleaching during long imaging runs.

Light Sheet Fluorescent Microscopy (LSFM) is a revolutionary technique that addresses all of these issues. Despite these advances, LSFM has not been widely adopted by plant scientists, as they did not contain the environmental conditions necessary to maintain plants within the machine. We have worked with Zeiss to specify a new sample chamber incorporating both temperature regulation and illumination where plants can be grown for several days. We represent a group of 3 institutions within the Midlands that propose to establish the first LSFM facility in the UK and one of the first in the world that has been specifically optimized for plant research. We will encourage users from throughout the Midlands and beyond to access this facility. Our consortium represents investigators from a variety of different disciplines, bioscience, computer science, engineering and mathematics. Therefore, such a microscope would not only provide a step change to bioscientists, but also fuel the development of new software tools and mathematical models that will have a huge benefit to researchers throughout the UK.

The main beneficiaries of this investment in a Zeiss Z.1 LSFM that has been optimized for plant growth would be our user pool. Currently, this centers around plant groups from several institutions within the Midlands, but we anticipate users from further afield due to our unique lighting chamber. The research preformed with this microscope will not be limited to plants and we have groups working with Drosophila, zebrafish and in clinical science have all expressed interest in using this machine.

We have previously received a multimillion pound investment for plant phenotyping at the whole organ level from the European Research Council and the University of Nottingham. The LSFM would extend this, to allow the incorporation of phenotyping at the cell-scale. Provision of a light sheet microscope will support development of new bioimage analysis methods and associated software tools. Continuing established practice, these tools will be made freely available to the wider community under an Open Source license via the CPIB website (www.cpib.ac.uk) and SourceForge. Previous software tools produced by the Centre have been downloaded over 2000 times to date, and are widely cited. CPIB was recently invited to submit an Expression of Interest in becoming a Software Node of BioimagingUK. Access to a Iight sheet microscope would allow the Centre to develop a wider range of tools within BioimagingUK, significantly increasing the impact of Alert funding throughout the UK.

Plant Breeding Companies will be specifically interested in the plant phenotyping capabilities of this machine, especially related to analysis of complex 3-D structures such as anthers, as well as the novel molecular networks uncovered that affect agriculturally important traits such as root branching.

New companies involved in synthetic biology will be interested in the 3-D templates and advances in the multiscale modeling of biological systems that emerge from these projects.

The scientific community will benefit from both the research performed on this machine and on the image analysis tools developed. Scientists will be updated of new results through publications and presentations at conferences.

The community will benefit through a series of outreach events coordinated by our dedicated Outreach Officer, Susie Lydon. These involve work with schools, scientific shows and web-based dissemination of images.

A next generation of scientists will benefit through the training of young scientists using the microscope. We estimate that within the first year we will train approximately 30 graduate and postgraduate students. In addition the images produce will feed into our image analysis summer schools where we have so far trained an additional 50 participants. In addition we propose to work together with Aston University to set up an annual one day workshop for LSFM users, where we will discuss new techniques, best practices and advances in image analysis.