Danforth Center: A passion for plants
Image: Danforth's new X-ray Computed Tomography system is the first of its kind in US academic research.
Founded in 1998, the Donald Danforth Plant Science Center is a not-for-profit scientific facility located in St Louis, Missouri, United States, with a mission to "improve the human condition through plant science".
Widely-known in the plant science community as a world-class facility, twenty research teams are currently conducting basic research that is intended to improve agricultural productivity and preserve natural resources.
Tasked with reducing the need for pesticides and fertilizers, increasing the nutritional content of crops and improving resistance to drought, pests and disease, the researchers have access to myriad analysis technologies. These include specialised microscopes, 3D imaging platforms, mass spectrometers and state-of-the-art plant growth facilities.
Dr Howard Berg heads up the Integrated Microscopy Facility at the Center. When he established the Facility in 2001, his aim was to provide researchers with the best instruments to image plant cells and better understand cell biology.
Today, as Director of the core facility, he has a host of microscopes at his fingertips, including a Leica SP8-X confocal, a Zeiss PALM laser microdissection microscope, a Hitachi tabletop SEM and LEO 912 AB TEM.
Not surprisingly research has been varied, with current projects including RNA processing in maize anthers and how plant defensin peptides fight infection in cells.
Confocal Microscopy: Sections of rice leaf tissue used to analyze chloroplast (red) number and size in a genetic study of factors affecting bundle sheath cell chloroplasts. [Danforth]
But as Berg points out, a lot of his facility's research hinges on confocal microscopy, with many researchers using its Leica instrument.
"We acquired this instrument in 2013, following a Major Research Instrumentation grant from the National Science Foundation," he highlights. "The committee evaluating the options at the time, were unanimous that this offered us the best options."
For example, given the need for live cell imaging, the instrument's sensitive hybrid detectors - HyD - were a huge draw. According to Berg, a lot of research is now directed at crop plants in the 'real world', rather than the widely-studied model plant, Arabidopsis, making highly sensitive detectors imperative.
"We have an inherently dimmer signal [with the crops] but have been able to orient the design features of our Leica for live cell imaging and a low signal," says Berg.
Meanwhile, the instrument's white laser light produces 200 lines, offering versatility for fluorescence imaging, especially, as Berg points out, when coupled with the sliding mirrors for selecting bandwidth detection.
"We also have a fast resonance scanner so we can do time-lapse imaging and preserve cell viability," he says.
"And the HyD detectors also have time gating so you can adjust the detection window of decay of a fluorophore," he adds. "This means we can eliminate auto-fluorescent compounds; plants are loaded with these compounds."
Berg currently trains students and postdoctoral researchers at Danforth and beyond, with his courses covering many areas of light microscopy, including advanced techniques, as well as hands-on experience of the Leica SP8-X.
With a background in plant cell structure, Dr Howard Berg helps to design and implement imaging experiments at the Danforth Center. [Danforth]
"I would, if I could, add a two-photon laser [to the microscope] for deeper penetration into plant tissue," highlights Berg. "Scattering with continuous wave lasers doesn't let us peer deep enough inside tissues."
Confocal microscopy aside, many researchers also use the facility's Zeiss PALM system to capture tissue components.
Here, the microscope's focused laser cuts out and isolates selected specimens without contact. And, as such, many researchers have exploited this to survey plants for expression of fluorescent protein constructs.
"We have an investigator that is looking at the abscission zone in setaria leaf in fluorescence, so she's capturing that region of tissue to look at gene expression," highlights Berg.
"In the past we have used the system for investigating parasitic feeding sites from root-knot nematodes," he adds. "These [sites] are very large regions, and the idea is to capture the giant cells that the nematode induces."
Crucially, the facility also integrates light microscopy with TEM; specifically the LEO 120 kV TEM - which, according to Berg, has provided so much useful information over the years.
TEM of nitrogen fixing bacteria in a root nodule of soybean
His facility is also home to a Bal-Tec HPM 010 High Pressure Freezing Machine for sample preparation, which according to Berg produces 'wonderful material' for thin sectioning.
"I wouldn't do TEM without these high-pressure frozen samples as the fidelity and preservation is astounding," he says.
"Now researchers are realizing that they need even more detail, TEM is back on the upsurge again for biology research, which is great," he adds. "Even with super-resolution microscopy you can't see the context of the fluorophore, yet with TEM you see everything."
But it's not all about high resolution. Investigators also use a Hitachi TM-1000 tabletop SEM for measuring cell size within plant tissue as well as looking at phenotypes of plant surface structures.
And Berg and colleagues have also been using the open access platform - OpenSPIM - to build a light sheet microscope. "Light sheet microscopy is a good way to study, for example, the development of Volvox, as it has such a large volume," says Berg.
A little over a year ago, the Danforth Plant Science Center also installed an industrial-scale North Star Imaging X5000 X-ray Computed Tomography system.
Managed by research scientist, Keith Duncan, the system is the first of its kind in US academic research dedicated to plant science, and provides insight into root systems and plant growth.
As Duncan points out, studies to date have largely focused on research with Valent Biosciences, a major collaboration partner on the X-ray CT system, examining the root systems of numerous crops and interactions with rhizosphere organisms.
As part of a National Science Foundation grant, Duncan and colleagues have also been scanning a large maize breeding population using root system architecture to explore genetic traits that could reveal new breeding strategies.
3D volume of a mature grain sorghum panicle imaged : a helical X-ray CT scan collected 18,000 digital radiographs which were compiled into a single 3D volume, allowing digital non-destructive analysis of the entire panicle [Danforth]
"Any aspect of root growth is going to be crucially important and being able to see the roots is just tremendous," says Duncan. "But we also have colleagues looking at a nearly endless variety of biological questions... including storage root production in cassava, ear and tassel development on sorghum and wax deposition on the moss, physcomitrella."
"You can't emphasise how unique it is having this instrument here at the Danforth Center," adds Duncan. "And the joke is, anything I can fit into the instrument, I will scan."
Duncan and colleagues are also collaborating with researchers at the University of Washington, to combine X-ray CT with positron emission tomography and track exactly how specific elements travel from plant leaves to roots.
Duncan reckons magnetic resonance imaging and nuclear magnetic resonance are potential complementary technologies.
"These techniques allow you to track actual dynamic movement, so for example, we could also follow fluid dynamics," he explains.
Confocal microscopy: Autofluorescence in a transverse section of tomato leaf. [Danforth]
The research scientist also hopes that his facility will soon acquire a X-ray micro-CT unit, to study smaller samples at higher resolution than the industrial-scale X-ray CT system.
But right now, the Danforth Center is the only facility in the US with an industrial-scale X-ray instrument dedicated exclusively to plant biology. Duncan doesn't expect this to last for long.
"There is this small community of X-ray imaging specialists in plant science; for example, the Hounsfield facility at the University of Nottingham, UK, has being doing this for years and we have modelled ourselves on this," he says. "But word is getting out and twelve months from now I anticipate there will be at least six more instruments across North America."
"People are simply starting to see the results and they're starting to see specifically what you can do when you have time to spend with X-ray CT and have it available to augment your research programme on a day-to-day basis," he adds.