The authors would like to thank S Castorani, AJ Eustis, GA Gambetta, CM Manuck, Z Nasafi, and A Zedan. This work was funded by: the U.S. Department of Agriculture-Agricultural Research Service Current Research Information System funding (research project no. 5306-21220-004-00; The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.); and NIFA Specialty crops research initiative grant to AJM.

Protocol details described below were written specifically for work at the Advanced Light Source 8.3.2 beamline. Adaptations may be required for work at other synchrotron facilities. Proper safety and radiation training is required for use of these facilities.
1. Sample Preparation for Live Plants
<ol>
 <li>Grow plants in ~10 cm diameter pots, and ensure that the main stem (or portion of the plant to be scanned) is as centered as possible and oriented vertically in the pot. The phys...

<ol>
	<li>Brodersen, C. R., McElrone, A. J., Choat, B., Matthews, M. A., Shackel, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+embolism+repair+in+xylem:+in+vivo+visualizations+using+high+resolution+computed+tomography.">The dynamics of embolism repair in xylem: in vivo visualizations using high resolution computed tomography.</a> Plant Physiology. 154, 1088-1095 (2010).</li><li>Brodersen, C. R., Lee, E., Choat, B., Jansen, S., Phillips, R. J., Shackel, K. A., McElrone, A. J., Matthews, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+analysis+of+3D+xylem+networks+using+high+resolution+computed+tomography+(HRCT).">Automated analysis of 3D xylem networks using high resolution computed tomography (HRCT).</a> New Phytologist. 191 (4), 1168-1179 (2011).</li><li>Brodersen, C., Roark, L., Pittermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+implications+of+primary+xylem+organization+in+two+ferns.">The physiological implications of primary xylem organization in two ferns.</a> Plant, Cell &amp; Environment. , (2012).</li><li>Brodersen, C., Choat, B., Chatelet, D., Shackel, K. A., Matthews, M. A., McElrone, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conductive+xylem+bridges+contribute+differentially+to+radial+connectivity+in+grapevine+stems+(Vitis+vinifera+and+V.+arizonica).">Conductive xylem bridges contribute differentially to radial connectivity in grapevine stems (Vitis vinifera and V. arizonica).</a> American Journal of Botany. , (2012).</li><li>McElrone, A. J., Jackson, S., Habdas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydraulic+disruption+and+passive+migration+by+a+bacterial+pathogen+in+oak+tree+xylem.">Hydraulic disruption and passive migration by a bacterial pathogen in oak tree xylem.</a> Journal of Experimental Botany. 59, 2649-2657 (2008).</li><li>McElrone, A. J., Grant, J., Kluepfel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ethylene-induced+tyloses+in+canopy+hydraulic+failure+of+mature+walnut+trees+afflicted+with+apoplexy+disorder.">The role of ethylene-induced tyloses in canopy hydraulic failure of mature walnut trees afflicted with apoplexy disorder.</a> Tree Physiology. 30, 761-772 (2010).</li><li>Tyree, M., Sperry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vulnerability+of+xylem+to+cavitation+and+embolism.">Vulnerability of xylem to cavitation and embolism.</a> Annual Review of Plant Biology. 40 (1), 19-36 (1989).</li><li>Tyree, M., Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xylem structure and the ascent of sap. , (2002).</li><li>Holbrook, N. M., Zwienieck, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vascular Transport in Plants. , (2005).</li><li>Mayo, S. C., Chen, F., Evans, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micron-scale+3D+imaging+of+wood+and+plant+microstructure+using+high-resolution+x-ray+phase-contrast+microtomography.">Micron-scale 3D imaging of wood and plant microstructure using high-resolution x-ray phase-contrast microtomography.</a> Journal of Structural Biology. 171, 182-188 (2010).</li><li>Mannes, D., Marone, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+areas+of+synchrotron+radiation+tomographic+microscopy+for+wood+research.">Application areas of synchrotron radiation tomographic microscopy for wood research.</a> Wood Science and Technology. 44, 67-84 (2010).</li><li>Maeda, E., Miyake, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-destructive+tracing+with+an+x-ray+micro+ct+scanner+of+vascular+bundles+in+the+ear+axes+at+the+base+of+the+lower+level+rachis-branches+in+japonica+type+rice+(oryza+sativa.">A non-destructive tracing with an x-ray micro ct scanner of vascular bundles in the ear axes at the base of the lower level rachis-branches in japonica type rice (oryza sativa.</a> Japanese Journal of Crop Science. 78 (3), 382-386 (2009).</li><li>Steppe, K., Cnudde, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+x-ray+computed+microtomography+for+non-invasive+determination+of+wood+anatomical+characteristics.">Use of x-ray computed microtomography for non-invasive determination of wood anatomical characteristics.</a> Journal of Structural Biology. 148 (1), 11-21 (2004).</li><li>Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicotyledonous+wood+structure+(made+apparent+by+sequential+sections).">Dicotyledonous wood structure (made apparent by sequential sections).</a> Encyclopaedia Cinematographica. , (1971).</li><li>Lee, E. F., Brodersen, C. R., McElrone, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+HRCT-derived+xylem+network+reveals+reverse+flow+in+some+vessels.">Analysis of HRCT-derived xylem network reveals reverse flow in some vessels.</a> , (2013).</li><li>McDowell, N. G., Pockman, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+plant+survival+and+mortality+during+drought:+why+do+some+plants+survive+while+others+succumb.">Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb.</a> New Phytologist. 178, 719-739 (2008).</li><li>McElrone, A. J., Brodersen, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifuge+technique+consistently+overestimates+vulnerability+to+water-stress+induced+cavitation+in+grapevines+as+confirmed+with+high+resolution+computed+tomography.">Centrifuge technique consistently overestimates vulnerability to water-stress induced cavitation in grapevines as confirmed with high resolution computed tomography.</a> New Phytologist. , (2012).</li><li>Lee, K., Avondo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+plant+development+and+gene+expression+in+three+dimensions+using+optical+projection+tomography.">Visualizing plant development and gene expression in three dimensions using optical projection tomography.</a> Plant Cell. 18, 2145-2156 (2006).</li><li>Truernit, E., Bauby, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+whole-mount+imaging+of+three-dimensional+tissue+organization+and+gene+expression+enables+the+study+of+phloem+development+and+structure+in+Arabidopsis.">High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis.</a> Plant Cell. 20, 1494-1503 (2008).</li><li>Jahnke, S., Menzel, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+MRI-PET+dissects+dynamic+changes+in+plant+structures+and+functions.">Combined MRI-PET dissects dynamic changes in plant structures and functions.</a> The Plant Journal. 59, 634-644 (2009).</li><li>Iyer-Pascuzzi, A. S., Symonova, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+analysis+platform+for+automatic+phenotyping+and+trait+ranking+of+plant+root+systems.">Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems.</a> , (2010).</li></ol>

Synchotron HRCT provides plant biologists with a powerful, non-destructive tool to explore the inner workings of plant vasculature in incredible detail. This technology has been used recently to identify previously undescribed anatomical structures in grapevine xylem that differentially alter xylem network connectivity in various grapevine species (Brodersen et al. 2012b, in press)- this connectivity can drastically alter the ability of vascular pathogens and emboli to spread destructively throughout xyle...

Water is transported from plant roots to the leaves in a vascular tissue called xylem - a network of interconnected conduits, fibers, and living, metabolically active cells. Transport function of plant xylem must be maintained to supply nutrients and water to leaves for photosynthesis, growth, and ultimately survival. Water transport in xylem conduits can be disrupted when the xylem network is compromised by pathogenic organisms. In response to such infections plants often produce gels, gums, and tyloses as a means to isolate pathogen spread (e.g. McElrone et al 2008; 2010). Drought stress can also limit water transport in xylem. As plants lose water during prolonged drought, tension builds in the xylem sap. Water under tension is metastable (i.e. at a certain threshold the tension becomes great enough to cavitate water columns contained in xylem conduits). After cavitation occurs, a gas bubble (embolism) can form and fill the conduit, effectively blocking water movement (Tyree and Sperry 1989), a phenomenon analogous to decompression sickness (i.e. "the bends") in deep sea divers.
Despite the importance of xylem water transport for optimal plant function as demonstrated by a vast body of historical and contemporary literature on this topic (Tyree &amp; Zimmermann, 2002; Holbrook et al., 2005), there are still aspects of xylem networks that remain elusive. Several research groups have recently begun utilizing High resolution x-ray computed micro-tomography (HRCT) to evaluate finer details of wood anatomy and vascular tissue (e.g. Mayo et al; 2010, 2008; Mannes et al. 2010; Brodersen et al. 2010, 2011, 2012a,b; Maeda and Miyake, 2009; Steppe et al. 2004). HRCT is a nondestructive technique used to visualize features in the interior of solid objects and to obtain digital information on their 3-D structural properties. HRCT differs from conventional medical CAT-scanning in its ability to resolve details as small as a micron in size, even for high density objects. Recent advances in synchrotron HRCT technology have improved image resolution and signal to noise ratio sufficiently so that plant vessel networks and intervessel connections can be visualized, assigned 3D coordinates, and exported for hydraulic model simulations. Brodersen et al. (2011) recently advanced this technique by combining 3D reconstructions generated by synchrotron HRCT with a Fortran model that automatically extracts data from the xylem network at much higher resolution than was ever possible with traditional anatomical methods (i.e. serial sectioning with a microtome and image capture with light microscopy, e.g. Zimmermann 1971). This work has also been used to optimize hydraulic models of the xylem system and identified unique characteristics of transport (i.e. reverse flow in some vessels during periods of peak transpiration) (Lee et al., in review).
Synchrotron HRCT can now be used to visualize xylem functionality, susceptibility to cavitation, and a plants' ability to repair embolized conduits. Failure to re-establish flow in embolized conduits reduces hydraulic capacity, limits photosynthesis, and results in plant death in extreme cases (McDowell et al. 2008). Plants can cope with emboli by diverting water around blockages via pits connecting adjacent functional conduits, and by growing new xylem to replace lost hydraulic capacity. Some plants possess the ability to repair breaks in the water columns, but the details of this process in xylem under tension have remained unclear for decades. Brodersen et al. (2010) recently visualized and quantified the refilling process in live grapevines using HRCT. Successful vessel refilling was dependent on water influx from living cells surrounding the xylem conduits, where individual water droplets expanded over time, filled vessels, and forced the dissolution of entrapped gas. The capacity of different plants to repair compromised xylem vessels and the mechanisms controlling these repairs are currently being investigated.
Description of the ALS facility Beamline 8.3.2
Our work to date has been conducted on the Hard X-ray Micro-Tomography Beamline 8.3.2 at the Advanced Light Source in Lawrence Berkeley National Lab (Berkeley CA USA). Plant samples are placed in a lead-lined hutch located 20 m from the x-ray source, generated by a 6 Tesla superconducting bend magnet dipole within the Advanced Light Source electron storage ring operating at a critical energy of 11.5 KeV. A schematic of the end station is shown in Figure 1. The x-rays enter the hutch with a beam size of 40x ~4.6 mm and pass through the sample that is mounted on a motorized rotating stage. The transmitted x-rays impinge on a crystal scintillator (two materials commonly used are LuAG or CdWO4) which convert x-rays to visible light that is relayed via lenses onto a ccd for image collection. The camera, scintillator and optics are contained in a light tight box that is on rails that allows the sample-to-scintillator distance to be optimized for phase contrast imaging.
All samples are mounted on the 10 cm diameter rotary stage which in turn is mounted on horizontal and vertical translation stages for sample positioning. A living plant sample, with the root system mounted in a custom built plant pot holder and the foliage contained in an acrylic tube, can be seen in Figure 2. Typical exposure times can range from 0.1- 1 sec using 10-18 KeV, and scan durations will range from 5-40 min depending on the settings optimized for a particular sample. For tall samples (typical of plant xylem networks), data scans can be tiled by repeating the measurement with the sample at different heights, which is controlled automatically, allowing seamless serial sections along a maximum sample height of ~ 10 cm. Maximum sample width when imaging at 4.5 &mu;m resolution is ~1 cm for samples that are nearly perfect in vertical orientation. Data generation and processing is completed using the protocol listed below. Because of the difference in x-ray attenuation between air and water, excellent image contrast can be obtained in plants without the use of contrast solutions typical of medical CT systems. The air-filled vessel lumen is easily distinguishable from the surrounding water-filled tissue in hydrated plants.

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using high resolution computed tomography to visualize the three dimensional structure and function of plant vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

Synchotron HRCT scans have been successfully implemented on a wide variety of plant tissues and species using beamline 8.3.2 (Figure 5), and have provided new insights into the structure and function of plant xylem at unprecedented resolution in 3D. The visualization and exploration capabilities provided by the 3D reconstructions (as illustrated in Figures 6-8; and Movies 1-3) allow for precise determination of location and orientation of structures with the xylem networks on both...

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Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

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20.9K 조회수.  U.S. Department of Agriculture. 고해상도 X 선 계산 단층 촬영 (HRCT)는 3D로 공장 vasculature의 구조와 기능을 연구하는 데 사용할 수있는 비파괴 진단 이미징 기술이다. 우리는 식물 조직 및 종의 다양한 범위에 걸쳐 목부 네트워크 탐색을 용이하게하는 방법 HRCT 보여줍니다.