High Throughput Fluorescence Imaging for Plant Sciences

Lead Research Organisation: University of Oxford
Department Name: Plant Sciences

Abstract

Research cell and developmental biology relies on imaging structures and molecules within living cells. In this way biological process can be followed in 3D over time scales ranging from tens of milliseconds to tens of hours. The most widely applied method for collecting such images is confocal fluorescence microscopy which provides clear images from within living tissues without the need to kill and physically section the specimen. The effectiveness of the method depends, first, on the ability to tag specific molecules with a fluorescent label and, second, on the ability of the microscope to form an image with high resolution and contrast whilst excluding out-of-focus information. Recent advances in structured illumination techniques, such as the Zeiss apotome system, have allowed optical sectioning at lower-magnifications which greatly facilitates initial fluorescence screening prior to confocal imaging, or measurements of physiological and developmental processes operating at much larger spatial scales (several cm squared) needed to image entire intact plant tissues and organs. Thus the ideal solution to be able to track developmental and physiological responses needs to combine low-magnification fluorescence for initial screening with high-resolution follow-up for detailed cellular and sub-cellular resolution. The addition of a robotic system tailored to the demands of plant specimens maximises the efficiency of data collection needed to achieve high-throughput for screening projects, or to allow long-term unattended operation for developmental studies. Such automated high-throughput fluorescence screening is routine in animal studies, where cells can be easily grown in multi-well culture plates, but a pipeline specifically tailored to handle the range of spatial scales and developmental time periods appropriate for plant systems would be unique in the UK.

Technical Summary

The use of fluorescent reporters and live-cell imaging has revolutionised our ability to probe plant and fungal development and physiology in unprecedented detail. The latest generation of microscopes are sufficiently sensitive to visualise responses in three (x,y,z) and four (x,y,z,t) dimensions for multiple probes at different wavelengths over a wide range of temporal and spatial scales to determine how plants grow and respond to their environment. However, operating these microscopes is time-consuming and requires considerable expertise to collect good quality data, particularly from plant tissues, often limiting the throughput of the instruments, and the type of research questions that can be tackled within a reasonable time frame. Thus, we have developed an automated fluorescence imaging system specifically tailored to the demands of plant systems that comprises a low-magnification Zeiss AxioZoom V16 fluorescence microscope as the front-end, that is fed samples in a variety of plate formats using a Peak Analysis and Automation (PAA) KiNEDx robotic automation system, with specimens maintained in a custom-built illuminated carousel growth incubator. The robot also interfaces directly to a Zeiss LSM 880 AiryScan confocal system for high-resolution follow-up measurements for regions-of-interest (ROIs), where co-ordinates are automatically transferred within the Overlord software environment. Both microscopes are equipped with motorized sample stages to allow registration and seamless integration of data collected at low magnification to provide context, with images collected at high-resolution to provide detail. The system can operate in three modes to allow high-throughput automated screening of reporter lines, repeated sampling over extended time periods for developmental studies at low and high spatial resolution, and both multiplexed, wide-field of view physiological studies to complement cell-specific high-resolution measurements.

Planned Impact

The world-class research that is supported by the microscopy facility in the Department of Plant Sciences is focused on providing a deep understanding of fundamental plant and microbial biology and is disseminated through major academic publications and conference publications. This fundamental research also underpins patent applications, technology transfer and supports spin-out companies to commercialise applications of the research.

For example research into chloroplast biogenesis has led to new opportunities to protect plants against the sorts of environmental stresses that are likely to become increasingly problematic in agriculture as climates change ( Jarvis, P. (2016) Plant discovery could help develop stress-resistant crops. BBSRC Business Winter 2016, p. 17.; http://www.bbsrc.ac.uk/news/food-security/2015/150918-pr-plant-discovery-help-develop-stress-resistant-crops/). This and research into the evolution of rooting structures (Dolan) in the earliest land plants has resulted in Follow-On Funding for commercialisation of research findings and similar strategies are being followed by other users of the facility, though details are commercially sensitive.

Other work supported by the facility in the Moore lab has provided unexpected insights into the cellular function of a protein involved in human neurodegenerative disease (Of Axons and Root Hairs: Plants in the Neurodegeneration Lab? http://www.alzforum.org/news/research-news/axons-and-root-hairs-plants-neurodegeneration-lab).

The facility also has impact in the commercial sector, supporting the R&D activities of the University spin-out company, Oxford Nanopore Technologies Ltd over several years, in the development of their biological analysis tools such as the worlds first and only nanopore DNA sequencer, the MinION. Other users of the microscope based in Engineering Science use the microscopy facility study bacterial biofilms and their impact on water quality and industrial processes.

Finally, the facility is used regularly for outreach and University access activities with members of the public or prospective students who may not consider applying to university or pursuing a career in plant biology. The images that are acquired also form the basis of public engagement activities in local museums, botanic gardens, and further afield and provide insight into a hidden world of cellular structure and dynamics rarely fails to impress.