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.
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.
Organisations
Publications
Jaeger R
(2021)
A fundamental developmental transition in Physcomitrium patens is regulated by evolutionarily conserved mechanisms
in Evolution & Development
Durr J
(2021)
A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.
in Plant & cell physiology
Sandor A
(2024)
Characterization of intracellular membrane structures derived from a massive expansion of endoplasmic reticulum (ER) membrane due to synthetic ER-membrane-resident polyproteins.
in Journal of experimental botany
Serra L
(2022)
Flip-Flap: A Simple Dual-View Imaging Method for 3D Reconstruction of Thick Plant Samples.
in Plants (Basel, Switzerland)
Soldan R
(2021)
From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization.
in Environmental microbiology
Moody LA
(2018)
Genetic Regulation of the 2D to 3D Growth Transition in the Moss Physcomitrella patens.
in Current biology : CB
Kalde M
(2019)
Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.
in The Plant journal : for cell and molecular biology
Sandor A
(2021)
IntEResting structures: formation and applications of organized smooth endoplasmic reticulum in plant cells
in Plant Physiology
Rutten PJ
(2021)
Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis.
in PLoS genetics
Aguilar-Trigueros C
(2022)
Network traits predict ecological strategies in fungi
in ISME Communications
Moody LA
(2021)
NO GAMETOPHORES 2 Is a Novel Regulator of the 2D to 3D Growth Transition in the Moss Physcomitrella patens.
in Current biology : CB
Pain C
(2019)
Quantitative analysis of plant ER architecture and dynamics.
in Nature communications
Spatola Rossi T
(2023)
Recombinant expression and subcellular targeting of the particulate methane monooxygenase (pMMO) protein components in plants.
in Scientific reports
Hughes T
(2020)
SCARECROW gene function is required for photosynthetic development in maize
in Plant Direct
Moody LA
(2018)
Somatic hybridization provides segregating populations for the identification of causative mutations in sterile mutants of the moss Physcomitrella patens.
in The New phytologist
Hoehne MN
(2022)
Spatial and temporal control of mitochondrial H2 O2 release in intact human cells.
in The EMBO journal
Elliott L
(2020)
Spatio-temporal control of post-Golgi exocytic trafficking in plants.
in Journal of cell science
Moreno-Ruiz D
(2021)
Stress-Activated Protein Kinase Signalling Regulates Mycoparasitic Hyphal-Hyphal Interactions in Trichoderma atroviride.
in Journal of fungi (Basel, Switzerland)
ELLIOTT L
(2019)
The importance of being edgy: cell geometric edges as an emerging polar domain in plant cells
in Journal of Microscopy
Mendes MA
(2020)
The RNA-dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in Arabidopsis.
in Development (Cambridge, England)
Garcia V
(2020)
TRIPP Is a Plant-Specific Component of the Arabidopsis TRAPPII Membrane Trafficking Complex with Important Roles in Plant Development
in The Plant Cell
Description | The grant was for development of high-throughput fluorescence imaging in plant sciences and has contributed to numerous projects within the department. The msot notable published work is automated quantitation of the sub-cellular architecture and dynamics of the plant endoplasmic reticulum. This provides imaging protocols and a complete software packaged developed using the equipment that is available on-line for other scientist to use in their research. Other projects include time-lapse imaging of root colonisation by N2-fixing bacteria, imaging 3D growth development in plants, and extended, time-lapse quantitation of fungal mycelial networks and functional traits. |
Exploitation Route | The equipment is already widely used by researchers in Plant Sciences and also Zoology. The general strategy for the facility also includes making all software and protocols available on-line for other users. |
Sectors | Agriculture, Food and Drink |
Title | AnalyzER : Quantitative analysis of plant ER architecture and dynamics. |
Description | The AnalyzER program is designed to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. It automatically quantifies: (i) The length, width, morphology and protein distribution along ER tubules; (ii) The degree and branch angles at junctions (nodes) in the tubular network; (iii) The size, shape, and protein distribution in cisternae and around the perimeter of the cisternae; (iv) The topological organisation of the tubular and cisternal network determined using graph-theoretic metrics; (v) The distribution of immobile nodes, tubules and cisternae using persistency mapping; (vi) The local speed, direction, coherence, divergence and curl of movement of tubules and cisternae using optical flow; and (vii) The size and shape of the polygonal regions enclosed by the network. |
Type Of Technology | Software |
Year Produced | 2018 |
Open Source License? | Yes |
Impact | Publication in press in Nature communications |
URL | https://ora.ox.ac.uk/objects/uuid:cb0e2845-2a9c-495a-84f0-4dd2c5164463 |