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Finding thresholds at which loss of plant functionality occurs during drought is critical for predicting future crop productivity and survival. Xylem resistance to embolism has been suggested as a key trait associated with water-stress tolerance. Although a substantial literature exists describing the vulnerability of woody stems to embolism, leaves and roots of herbaceous species remain under-represented. Also, little is known about vulnerability to embolism at a whole-plant scale or propagation of embolism within plants. New techniques to view the process of embolism formation provide opportunities to resolve long-standing questions. Here, we used multiple visual techniques, including X-ray microcomputed tomography and the optical vulnerability method, to investigate the spread of embolism within intact stems, leaves and roots of Solanum lycopersicum (common tomato). We found that roots, stems and leaves of tomato plants all exhibited similar vulnerability to embolism, suggesting that embolism rapidly propagates among tissues. Although we found scarce evidence for differentiation of xylem vulnerability among tissues at the scale of the whole plant, within a leaf the midrib embolized at higher water potentials than lower order veins. Substantial overlap between the onset of cavitation and incipient leaf damage suggests that cavitation represents a substantial damage to plants, but the point of lethal cavitation in this herbaceous species remains uncertain.

• Vulnerability curves (VCs) describe the loss of hydraulic conductance against increasing xylem tension, providing valuable insights about the response of plant water transport to water stress. Techniques to construct VCs have been developed and modified continuously, but controversies continue. • We compared VCs constructed using the bench-top dehydration (BD), air-injection-flow (AI), pneumatic-air-discharge (PAD), optical (OP) and X-ray-computed microtomography (MicroCT) methods for tropical trees and lianas with contrasting vessel lengths. • The PAD method generated highly vulnerable VCs, the AI method intermediate VCs, whereas the BD, OP and MicroCT methods produced comparable and more resistant VCs. Vessel-length and diameter accounted for the overestimation ratio of vulnerability estimated using the AI but not the PAD method. Compared with directly measured midday embolism levels, the PAD and AI methods substantially overestimated embolism, whereas the BD, MicroCT and OP methods provided more reasonable estimations. • Cut-open vessels, uncertainties in maximum air volume estimations, sample-length effects, tissue cracks and shrinkage together may impede the reliability of the PAD method. In conclusion, we validate the BD, OP and MicroCT methods for tropical plants, whereas the PAD and AI need further mechanistic testing. Therefore, applications of VCs in estimating plant responses to drought need to be cautious.

IntroductionDiffuse fibrosis is integral to many cardiomyopathies but is challenging to quantify and often relies on biopsy. Cardiac MRI has emerged as a non-invasive test to quantify extracellular volume fraction (ECV), a histologically validated surrogate of diffuse fibrosis. ECV can be calculated from pre- and post-gadolinium contrast T1 maps. However, cost, availability, scan duration and complexity of T1 measures limit their application. Contrast enhanced CT has been used to measure ECV in humans and may offer more rapid and reliable quantification, but this approach has yet to be applied in mice.1 MethodControl (n = 4) and infarcted (n = 4) BALB/C mice underwent microCT (PerkinElmer QuantumGX2, 90 kV, 88 uA, 72 mm voxel size) before and up to 15 minutes after intravenous infusion of Iohexol (Omnipaque) (figure 1). Regions of interest (ROIs) were drawn in the blood and septum. The mean Hounsfield units (HU) were measured and plotted against time. Haematocrit (Ht) was fixed at 45% (2). ECV was calculated as:ECVmyo = (1-Ht) x ([delta]HUmyo / [delta]HUblood)MicroCT ECV measurements were validated against gated MRI acquired at 9.4T (Agilent). T1 relaxation times were measured using a Look-Locker Inversion Recovery sequence pre- and 15 mins post- intraperitoneal injection of 0.5 mM Gd-DTPA.2 T1s from ROIs in the septum and blood pool were used to calculate ECV as:ECVmyo = (1-Ht) x ([delta](1/T1myo) / [delta](1/T1blood))ResultsMicroCT showed that equilibrium in HU was reached 10 minutes post contrast in healthy and infarcted mice (figure 2a, b, c). The mean ECV measured with microCT in healthy mice was 28% with a range of 22 ‘ 34%, and in infarcts was 29% with a range of 22 ‘ 35%. MRI ECV measurements had a mean of 21% with a range of 18 ‘ 24% in healthy mice, and 25% with a range of 20 ‘ 29% in infarcts. ECV measures correlated between modalities in healthy mice (r = 0.98, p = 0.019), infarcted mice (r = 0.98, p = 0.02), and when combined (r = 0.81, p = 0.013) (figure 2d). MicroCT overestimated ECV compared to MRI. Bland-Altman analysis in healthy and infarcted mice showed 5.65% bias with 95% confidence limits at -0.27% and 11.57% (figure 2e). These results demonstrate that microCT provides ECV measurements comparable with MRI in both healthy and infarcted mice. As expected,3 there was a trend towards increased ECV in the septum of infarcted mice.Abstract BS68 Figure 1MicroCT images of mouse hearts MicroCT images of a) a healthy mouse heart and b) an infarcted mouse heart, at diastole and systole pre- and post-contrast. Example ROIs in the septum and blood pool are shown in yellow. Area of myocardial infarction is indicated by white arrowsAbstract BS68 Figure 2MicroCT attenuation curves and ECV measurements MicroCT attenuation curves for blood and the myocardial septum of a) healthy mice and b) infarcted mice. Images acquired pre- (-5 minutes) and post- intravenous injection of Iohexol. c) MicroCT images of an infarcted mouse pre-contrast and up to 15 minutes post-contrast. d) Plot of ECV measured with microCT against MRI for healthy mice and infarcted mice (n = 8). e) Bland-Altman analysis comparing ECV measured by MRI and microCT of healthy and infarcted miceConclusionWe demonstrate the first use of microCT to measure ECV in infarcted mice with measures strongly correlating with established MRI T1 mapping. MicroCT has several advantages over MRI including, more rapid and 3D acquisitions; higher spatial resolution; and simpler analysis of HU. MicroCT offers an accurate and high-throughput method for preclinical efficacy testing of emerging antifibrotic agents which are urgently needed to treat heart disease.ReferencesBandula S, et al. Radiology. 2013.Stuckey D, et al. Circulation. 2014.Scully PR, et al. Curr Cardiol Rep. 2018.Conflict of InterestNone

Drought-induced xylem embolism is a serious threat to plant survival under future climate scenarios. Hence, accurate quantification of species-specific vulnerability to xylem embolism is a key to predict the impact of climate change on vegetation. Low-cost hydraulic measurements of embolism rate have been suggested to be prone to artefacts, thus requiring validation by direct visualization of the functional status of xylem conduits using nondestructive imaging techniques, such as X-ray microtomography (microCT). We measured the percentage loss of conductance (PLC) of excised stems of Laurus nobilis (laurel) dehydrated to different xylem pressures, and compared results with direct observation of gas-filled vs water-filled conduits at a synchrotron-based microCT facility using a phase contrast imaging modality. Theoretical PLC calculated on the basis of microCT observations in stems of laurel dehydrated to different xylem pressures overall were in agreement with hydraulic measurements, revealing that this species suffers a 50% loss of xylem hydraulic conductance at xylem pressures averaging −3.5 MPa. Our data support the validity of estimates of xylem vulnerability to embolism based on classical hydraulic techniques. We discuss possible causes of discrepancies between data gathered in this study and those of recent independent reports on laurel hydraulics.

Embolism removal is critical for restoring hydraulic pathways in some plants, as residual gas bubbles should expand when vessels are reconnected to the transpiration stream. Much of our understanding of embolism removal remains theoretical as a consequence of the lack of in vivo images of the process at high magnification. Here, we used in vivo X-ray micro-computed tomography (microCT) to visualize the final stages of xylem refilling in grapevine (Vitis vinifera) paired with scanning electron microscopy. Before refilling, vessel walls were covered with a surface film, but vessel perforation plate openings and intervessel pits were filled with air. Bubbles were removed from intervessel pits first, followed by bubbles within perforation plates, which hold the last volumes of air which eventually dissolve. Perforation plates were dimorphic, with more steeply angled scalariform plates in narrow diameter vessels, compared with the simple perforation plates in older secondary xylem, which may favor rapid refilling and compartmentalization of embolisms that occur in small vessels, while promoting high hydraulic conductivity in large vessels. Our study provides direct visual evidence of the spatial and temporal dynamics of the final stages of embolism removal.

Background: Recent advances in micro-computed tomography (MicroCT) imaging have enabled detailed investigations of human microvascular anatomy, providing new insights that may influence treatment options and optimize local reparative potential. This article describes a reproducible cadaveric perfusion technique for visualizing foot and ankle microvasculature using MicroCT, designed to support anatomical research and surgical planning studies. Methods: Ten matched pairs of fresh-frozen cadaveric lower limbs were used to develop this protocol. An 18-gauge angiocatheter was used to cannulate the anterior and posterior tibial arteries for perfusion of the foot and ankle, or the popliteal artery for perfusion of the entire lower leg. Clearing was performed sequentially with 0.9% saline, 3% hydrogen peroxide, and water. Perfusion was performed with a 50% barium sulfate/2.5% gelatin solution. Confirmatory images were obtained using mini c-arm fluoroscopy. Final images were obtained for microvascular assessment using a commercial MicroCT scanner. Integrity of the perfusate was visually evaluated on MicroCT over the course of 4 freeze-thaw cycles spanning 2 months. Results: All intraosseous and extraosseous microvascular structures were successfully visualized using MicroCT of the cadaveric lower extremities. Microvasculature was perfused in continuity without incidence of contrast extravasation. When present, intraosseous nutrient arteries of the first and fifth metatarsal, and branches of the tarsal sinus artery were appreciated. Contrast material remained visually consistent even after preforming surgical resections and undergoing multiple freeze-thaw cycles. Conclusion: This standardized perfusion technique was effective in the visualization of microvasculature in the foot and ankle. In addition to 3-dimensional mapping using MicroCT, this reproducible protocol can be used in numerous advanced imaging applications, including microvascular assessment following surgical reconstructions and instrumentation. Clinical Relevance: A refined understanding of the microvascular anatomy of the foot and ankle using MicroCT perfusion imaging can potentially guide surgical techniques to minimize iatrogenic injury and optimize healing. Visual Abstract This is a visual representation of the abstract.

Starch is the primary energy storage molecule used by most terrestrial plants to fuel respiration and growth during periods of limited to no photosynthesis, and its depletion can drive plant mortality. Destructive techniques at coarse spatial scales exist to quantify starch, but these techniques face methodological challenges that can lead to uncertainty about the lability of tissue-specific starch pools and their role in plant survival. Here, we demonstrate how X-ray microcomputed tomography (microCT) and a machine learning algorithm can be coupled to quantify plant starch content in vivo, repeatedly and nondestructively over time in grapevine stems (Vitis spp.). Starch content estimated for xylem axial and ray parenchyma cells from microCT images was correlated strongly with enzymatically measured bulk-tissue starch concentration on the same stems. After validating our machine learning algorithm, we then characterized the spatial distribution of starch concentration in living stems at micrometer resolution, and identified starch depletion in live plants under experimental conditions designed to halt photosynthesis and starch production, initiating the drawdown of stored starch pools. Using X-ray microCT technology for in vivo starch monitoring should enable novel research directed at resolving the spatial and temporal patterns of starch accumulation and depletion in woody plant species.

X-ray microtomography (microCT) is becoming a valuable noninvasive tool for advancing our understanding of plant–water relations. Laboratory-based microCT systems are becoming more affordable and provide better access than synchrotron facilities. However, some systems come at the cost of comparably lower signal quality and spatial resolution than synchrotron facilities. In this study, we evaluated laboratory-based X-ray microCT imaging as a tool to nondestructively analyse hydraulic vulnerability to drought-induced embolism in a woody plant species. We analysed the vulnerability to drought-induced embolism of benchtop-dehydrated Eucalyptus camaldulensis plants using microCT and hydraulic flow measurements on the same sample material, allowing us to directly compare the two methods. Additionally, we developed a quantitative procedure to improve microCT image analysis at limited resolution and accurately measure vessel lumens. Hydraulic measurements matched closely with microCT imaging of the current-year growth ring, with similar hydraulic conductivity and loss of conductivity due to xylem embolism. Optimized thresholding of vessel lumens during image analysis, based on a physiologically meaningful parameter (theoretical conductivity), allowed us to overcome common potential constraints of some lab-based systems. Our results indicate that estimates of vulnerability to embolism provided by microCT visualization agree well with those obtained from hydraulic measurements on the same sample material.

Water acquisition is thought to be limited to the unsuberized surface located close to root tips. However, there are recurring periods when the unsuberized surfaces are limited in woody root systems, and radial water uptake across the bark of woody roots might play an important physiological role in hydraulic functioning. Using X-ray microcomputed tomography (microCT) and hydraulic conductivity measurements (Lp r), we examined water uptake capacity of suberized woody roots in vivo and in excised samples. Bark hydration in grapevine woody roots occurred quickly upon exposure to water (c. 4 h). Lp r measurements through the bark of woody roots showed that it is permeable to water and becomes more so upon wetting. After bark hydration, microCT analysis showed that absorbed water was utilized to remove embolism locally, where c. 20% of root xylem vessels refilled completely within 15 h. Embolism removal did not occur in control roots without water. Water uptake through the bark of woody roots probably plays an important role when unsuberized tissue is scarce/absent, and would be particularly relevant following large irrigation events or in late winter when soils are saturated, re-establishing hydraulic functionality before bud break.

The mesophyll surface area exposed to intercellular air space per leaf area (S m) is closely associated with CO2 diffusion and photosynthetic rates. S m is typically estimated from two-dimensional (2D) leaf sections and corrected for the three-dimensional (3D) geometry of mesophyll cells, leading to potential differences between the estimated and actual cell surface area. Here, we examined how 2D methods used for estimating S m compare with 3D values obtained from high-resolution X-ray microcomputed tomography (microCT) for 23 plant species, with broad phylogenetic and anatomical coverage. Relative to 3D, uncorrected 2D S m estimates were, on average, 15–30% lower. Two of the four 2D S m methods typically fell within 10% of 3D values. For most species, only a few 2D slices were needed to accurately estimate S m within 10% of the whole leaf sample median. However, leaves with reticulate vein networks required more sections because of a more heterogeneous vein coverage across slices. These results provide the first comparison of the accuracy of 2D methods in estimating the complex 3D geometry of internal leaf surfaces. Because microCT is not readily available, we provide guidance for using standard light microscopy techniques, as well as recommending standardization of reporting S m values.