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 physical dimensions of the HRCT instrument hutch at the Advanced Light Source limits live plants to ~1 m in height. As a consequence, imaging of live plants is best performed on seedlings/saplings grown in small pots. Depending on the experiment, different soil types can be used to control soil moisture content (e.g. in drought experiments), and for some plants with flexible shoots (e.g. vines) longer shoots can be carefully tucked into the acrylic tube described below (see Figures 1 and 2).</li>
 <li>Mount the live potted plants in a custom-made rigid aluminum pot holder. The top plate height can be adjusted to accommodate a range of pot heights. The top of the plate is designed to align with the top of the soil surface, and the plant protrudes from the center of the two-part plate. The purpose of the pot holder is to ensure the plant stem is held firmly in place to minimize vibration or sample motion. Minimizing sample motion during a scan is essential.</li>
 <li>Once mounted in the holder, measure the stem water potential or leaf transpiration using a Scholander style pressure chamber or a clip-on leaf porometer, respectively, to determine the physiological status of the plant prior to scanning.</li>
 <li>Place a thin walled acrylic cylinder over the plant and on top of the aluminum plant holder and secure it in place with clay putty to stabilize the sample (Figure 2). Any vibration or movement of the upper foliage will be transmitted down the stem and cause the plant tissue within the scanned area to move, ultimately leading to image distortion. The cylinder is used to contain plant foliage and prevent plant leaves from rubbing against other pieces of equipment in the hutch that would result in vibrations during a scan. Additional plastic wrap, paper towels, and tape should be used to further minimize vibration and movement of plant parts (see problems associated with sample movement in Figure 4). To reduce its x-ray absorption (which can decrease the image quality at a given exposure time), the containing cylinder should have as thin walls as possible while maintaining sufficient rigidity to perform its function.</li>
 <li>Attach the custom pot holder to the air bearing stage and lock it (screw) into place between the x-ray source and the imaging sensor and camera equipment. Position the stem as vertical as possible and center on magnetic chuck base to ensure the sample stays in the field of view during rotation.</li>
</ol>
2. Sample Preparation for Fresh, Excised Plant Tissue
<ol>
 <li>Fresh plant material, typically stems or petioles, can be scanned after immediate removal from a live plant. If the intent of the experiment is to visualize the entirety of the xylem network, water within the vessels must be evacuated and replaced with air. To do this, mount the sample in a Scholander style pressure chamber and push compressed air or nitrogen through the sample at low pressure (&lt; 0.05 MPa) for approximately 5 min. Species will differ in the time required to evacuate the vessel network. If the intent is to evaluate the extent of embolism formation in the fresh plant tissue, then excise samples from the plant using a fresh razor blade and make the cuts under water.</li>
 <li>Wrap the sample in a layer of Parafilm to prevent desiccation during the scan.</li>
 <li>Mount the sample in a drill-chuck fixed to a metal plate that is screwed into the air bearing stage. Center and orient the sample vertically as described above to ensure the sample remains in the field of view.</li>
</ol>
3. Sample Preparation for Dried Woody Tissues
<ol>
 <li>For optimal tissue sample visualization and image contrast, it is necessary to slowly dehydrate the entire woody tissue sample. Cut samples to approximately 6 cm in length. Select samples that are as straight as possible in the targeted scan region and have a diameter of &le;1 cm.</li>
 <li>Place the woody tissue sample into a drying oven at low temperature to slowly dry the sample without causing any cracking or splitting of the tissue. This process is likely to differ between species and tissues. For woody stems, 12 hr in a 40 &deg;C oven is typically sufficient to provide excellent contrast without causing significant changes in the physical structure of the stem (see problems with rapid drying demonstrated in Figure 3).</li>
 <li>In some situations it is desirable to have a fiduciary marker within the sample such that subsequent dissection and visualization with scanning electron microscopy can be oriented to specific points in the HRCT image. To do this, affix a metal or glass bead or wire to the outside of the stem using Parafilm. Another method is to use a silicone resin (e.g. RTV-141, Bluestar Silicones, East Brunswick, NJ) that can be injected into a single xylem conduit (see examples in Brodersen et al 2010). Once hardened, the silicone resin is clearly visible in the sample and easily distinguished from the other air-filled vessels. Use this marker to precisely locate specific regions of the sample.</li>
 <li>Mount the sample in the drill chuck and center as described above.</li>
</ol>
4. Sample Preparation for Leaf Tissue for Two Dimensional (2D) Radiograms
<ol>
 <li>To visualize vessel contents in leaves in near-real-time, leaves can be scanned to produce a 2D radiogram, similar to a dental x-ray. Mount the leaf between two sheets of thin acrylic plastic, and secure the edges with clips. Then attach the mounted sample to a post-holder system and position the optical breadboard next to the imaging system and x-ray source.</li>
</ol>
5. Scanning the Sample in the 8.3.2 Hutch
<ol>
 <li>Decide the magnification that will work best for your application. ALS Beamline 8.3.2 has the capability to scan with lenses with magnifications of 2x, 5x, and 10x. These result in image pixel sizes of 4.5, 2.25, and 0.9 &mu;m, respectively. Depending on the magnification, the sample must be of appropriate size, as the field of view decreases with increasing magnification. See details for choice of camera and lens and the resultant image parameters in Table 1.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td> </td>
 <td colspan="2">PCO.4000 (4008x2672)</td>
 <td colspan="2">PCO.Edge (2560x2160) (Optique Peter)</td>
 </tr>
 <tr>
 <td>Lens</td>
 <td>pixel (&mu;m)</td>
 <td>field of view (mm)</td>
 <td>pixel (&mu;m)</td>
 <td>field of view (mm)</td>
 </tr>
 <tr>
 <td>10x</td>
 <td>0.9</td>
 <td>3.6</td>
 <td>0.65 (0.69)</td>
 <td>1.7 (1.7)</td>
 </tr>
 <tr>
 <td>5x (4x)</td>
 <td>1.8</td>
 <td>7.2</td>
 <td>1.3 (1.72)</td>
 <td>3.3 (4.4)</td>
 </tr>
 <tr>
 <td>2x</td>
 <td>4.5</td>
 <td>18</td>
 <td>3.25 (3.44)</td>
 <td>8.3 (8.8)</td>
 </tr>
 <tr>
 <td>1x</td>
 <td>9</td>
 <td>36</td>
 <td>6.5 (-)</td>
 <td>16.6 (-)</td>
 </tr>
 </tbody>
</table>
Table 1. Details regarding available cameras and lenses at ALS 8.3.2.
<ol start="2">
 <li>Set the x-ray energy to 15 keV. This has been shown to provide excellent image contrast for most plant applications (see Brodersen et al. 2010, 2011, 2012a,b). Exposure times are generally dependent on the thickness and density of the sample (and thus the magnification used) range between 100 and 1,000 msec. Longer exposure times (as long as detector pixels are not saturated) will generally lead to higher signal to noise ratio, but at the cost of increased scan times.</li>
 <li>Choose an angular increment that is appropriate for your application. Samples are rotated 180&deg; during a scan, and the number of images taken during the rotation can have a significant impact on size of the dataset, length of the scan interval, and final image quality, but there are generally diminishing returns in quality. Typical scans are performed at 0.25&deg; increments, yielding 721 images per scan. Decreasing the increment to 0.125&deg; results in better images for visualizing fine details, but yields 1,440 images and thus a much larger dataset (for a typical region of interest, this means ~10-30 GB of data vs. 5 GB). However, the signal to noise ratio is often improved and worth both the increased scan time and data size. Dry stems that are unlikely to deform/shrink during a scan can be subjected to longer intervals (smaller angular increment) without detriment. When imaging live plants, where biological processes (e.g. embolism repair) take place on short time scales, opting for the shorter scan intervals is preferable to limit potential damaging effects of x-ray radiation on this tissue- although this comes at a potential loss of image quality. Shorter scan intervals can be achieved using the Continuous Tomography setting during which the sample continuously rotates while the images are captured.</li>
 <li>For each scan, "bright field" and "dark field" images must be corrected. Bright field images are images without the sample in the beam. These are often collected before and after the scan of the sample by horizontally translating the sample. Dark fields are collected by closing the x-ray shutter&mdash;this measured the amount of signal the camera shows with no x-rays.</li>
</ol>
6. Data Processing
<ol>
 <li>Transfer the "raw" 2D .TIF images, which were exported from the acquisition computer to a file server, to a data processing computer. If the computer has sufficient RAM, the data can be copied to a so-called "RAM Drive" (a portion of the RAM appears as a hard drive on the computer). In this way the software does not have to access a spinning hard drive, which is comparatively slow compared to a solid state drive or flash memory. This step significantly reduces the amount of time required to process datasets.</li>
 <li>The images must be converted to a percent transmission scale. Beamline 8.3.2 has a custom background normalization plug-in that can be downloaded and used with the freely available software packages ImageJ or Fiji (<a href="http://fiji.sc/" target="_blank">http://fiji.sc/</a>). It subtracts the dark counts from the images and normalizes the sample images by the bright fields to yield images that show percent transmission. Load normalized images into the Octopus software package (<a href="http://www.inct.be/en/software/octopus" target="_blank">http://www.inct.be/en/software/octopus</a>) and "reconstruct" the 3D dataset from the 2D raw .TIF files using the designated processing steps (Normalize images, Ring removal, Sinogram creation, Parallel beam reconstruction). This process then yields a series of .TIF transverse (cross sectional) images composed of "voxels" (volumetric pixel elements), each with an x, y, z coordinate and intensity values representing the x-ray linear absorption coefficient.</li>
</ol>
7. Visualization
<ol>
 <li>Visualize the stack of images in one of a variety of software packages. Freeware (e.g. Drishti, <a href="http://anusf.anu.edu.au/Vizlab/drishti/index.shtml" target="_blank">http://anusf.anu.edu.au/Vizlab/drishti/index.shtml</a>) can be used to visualize volumes or individual or stacks of images (e.g. ImageJ or FIJI). Other software packages can be used for 3D visualization. Our research group uses the Avizo software package (<a href="http://www.vsg3d.com/avizo/overview" target="_blank">http://www.vsg3d.com/avizo/overview</a>), but others such as Amira (<a href="http://www.amira.com/" target="_blank">http://www.amira.com/</a>) and VGStudioMax (<a href="http://www.volumegraphics.com/" target="_blank">http://www.volumegraphics.com/</a>) are also commonly used.</li>
 <li>Load datasets into system memory and display the sample in virtual transverse, longitudinal, or radial slice orientations. Because of the 3D attributes of the dataset, virtual slices through the sample can be rotated in any plane to align with the regions of interest, a significant improvement over traditional serial light microscopy (see Movies 1-3 for detailed examples).</li>
 <li>To visualize the sample as needed in 3D, "segment" the sample using the variety of semi-automated and manual routines in Avizo to separate vessel lumens or other structures from the surrounding tissue. Segmentation refers to defining boundaries between objects of interest, thus separating or segmenting them into separate regions. Rendering volumes in 3D is performed by the visualization software. One method to do this is direct volume rendering, where each point in a volume is assumed to emit and absorb light; the amount and color of emission and absorption can be defined using a "colormap", and the resulting projection in a given direction is displayed on the screen. Alternatively, a wireframe or 3D mesh surface representing the segmented boundaries is constructed to show a 3D model of the structure of interest. The 3D mesh is composed of polygonal elements, and the total number of elements will affect both the fidelity of structure reproduction and the size of the associated data file (i.e. more elements leads to higher fidelity but larger file size). A variety of image processing modules are available within the visualization software to control the volume rendering outputs, as well as control for image brightness, contrast, transparency, noise reduction, etc.</li>
</ol>
8. Quantification
<ol>
 <li>Once segmentation has been accomplished, it is possible to quantify the target plant structures or functional changes in volume, length, width, presence or absence of water, air, etc. For example, Brodersen et al. (2010) used Avizo software to quantify the volume change of water droplets inside grapevine refilling vessels. Plants were scanned every 30 min over four to eight hours creating a time-lapse sequence of vessel refilling. Each scan was reconstructed and loaded into Avizo, where individual droplets were measured over time as their volume increased.</li>
</ol>

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We have nothing to disclose.

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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21.0K vistas.  U.S. Department of Agriculture. Alta resolución de rayos X de tomografía computarizada (HRCT) es una técnica no destructiva de imágenes diagnósticas que se pueden utilizar para estudiar la estructura y función de la vasculatura planta en 3D. Se demuestra cómo HRCT facilita la exploración de redes de xilema a través de una amplia gama de tejidos y especies vegetales.

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

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory&#039;s High Flux Isotope Reactor

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

Construction of a Realistic, Whole-Body, Three-Dimensional Equine Skeletal Model using Computed Tomography Data

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches have rarely been used to study equine athletic injury. Degenerative osteoarthritis caused by mechanical stress can originate from chronic postural dysfunction, which, because the primary dysfunction is often distant from the site of tissue injury, is best identified through modeling whole-body biomechanics. To characterize whole-body equine kinematics, a realistic skeletal model of a horse was created from equine computed tomography (CT) data that can be used for functional anatomical and biomechanical modeling. Equine CT data were reconstructed into individual three-dimensional (3D) data sets (i.e., bones) using 3D visualization software and assembled into a complete 3D skeletal model. The model was then rigged and animated using 3D animation and modeling software. The resulting 3D skeletal model can be used to characterize equine postures associated with degenerative tissue changes as well as to identify postures that reduce mechanical stress at the sites of tissue injury. In addition, when animated into 4D, the model can be used to demonstrate unhealthy and healthy skeletal movements and can be used to develop preventative and rehabilitative individualized therapies for horses with degenerative lamenesses. Although the model will soon be available for download, it is currently in a format that requires access to the 3D animation and modeling software, which has quite a learning curve for new users. This protocol will guide users in (1) developing such a model for any organism of interest and (2) using this specific equine model for their own research questions.

The purpose of this protocol is to describe the method of creation of a realistic, whole-body, skeletal model of a horse that can be used for functional anatomical and biomechanical modeling to characterize whole-body mechanics.

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches ...