Name: quantifying mixing using magnetic resonance imaging
Uploaded: 2012-01-25T12:00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about Quantifying Mixing using Magnetic Resonance Imaging at JoVE.com

The authors would like to thank Aspect Imaging (Industrial Area Hevel Modi'in, Shoham, Israel) for the pulse sequences used in the study. This work was partially funded by an award from the Center for Process Analytical Chemistry of the University of Washington (Seattle, WA, USA), as well as in-kind contributions and financial support from Procter and Gamble Company.

1. Mixer design 
<ol>
	<li>Use a CAD program to design the mixing sections of the static mixer.</li>
</ol>
The SAR mixer is composed of a number of different plate geometries; these geometries are shown in Fig. 3. Each laser-cut Poly (methyl methacrylate) (PMMA) plate is 1.59 mm thick and has a rectangular key at the bottom so that it can be aligned in a PVC pipe with an acrylic rod. The geometry is similar to those described in [3, 4], except that the walls in the expansions and contractions are formed by a series of discrete &ldquo;staircase&rdquo; steps due to the alignment of discrete plates rather than smooth diagonal surfaces. Although the material of construction here is PMMA and PVC, opaque non-metallic mixers can be constructed as well. 
<ol start="2">
	<li>Align the individual plates to develop the repeating units of the mixer. Position the plates tightly within a 1&frac12; inch Schedule 40 clear PVC pipe. </li>
	<li> Fig. 4 illustrates the static mixer viewed from the side. Notice that two fluids enter at the left side of the figure. The minor component, shown as the dark region, enters through the nozzle (Plate S, Fig. 3) and forms a stream of the minor component in the (colorless) major component. The repeating unit starts after Plate S at the first Plate C and extends downstream through Plate 48, which is also a Plate C. In each repeating unit, the two fluids flow into 8 plates of open channel (Plate C). The fluid is then physically separated into two vertical channels by eight plates of Plate I, followed by the actual mixing section. The mixing section is a total of 16 plates, from upstream to downstream: Plates I, A, B, D, E, F, G, J, J, K, L, M, N, O, P, and H. The fluid leaves the mixing section and flows into 8 plates of Plate H, in which fluid is physically split into two horizontal channels. The &ldquo;H&rdquo; section is followed by 8 plates of open channel (Plate C). This pattern of 48 plates is repeated 6 times in the mixer. Two repeating units are illustrated in Fig. 4 as Plates 1-96. </li>
</ol>
2. Flow system with MR imaging system and a mixer
<ol>
	<li>Assemble a flow system to pump Carbopol solution through the in-line split-and-recombine static mixer. Be able to control and record the mass flow rate of the test fluids. In addition, incorporate a pressure transducer upstream of the mixer to monitor pressure. </li>
	<li>Position the mixer in the magnet (Fig. 5). The magnet is part of a 1 Tesla permanent-magnet-based imaging spectrometer (Aspect Imaging, Industrial Area Hevel Modi'in, Shoham, Israel), with 0.3 T/m peak gradient strength The dimensions of the magnet enclosure are 700 x 700 x 600 mm.</li>
	<li>Dope part of the Carbopol solution with manganese chloride (MnCl2). This will be the minor component. The major component is undoped Carbopol solution. Fig. 6 illustrates a schematic of flow system. </li>
</ol>
3. Characterization of the test fluid 
<ol>
	<li>Prepare a 0.2 % w/w Carbopol (The Lubrizol Corporation) solution by slowly sifting a weighed amount of polymer into deionized water in a stirred tank. This polymer family of products is based on crosslinked acrylic acid chemistry and is widely used in personal care and household products as rheological modifiers. Neutralize the carbopol solution with a 50% NaOH solution to pH 7; the neutralization allows the solution to achieve its maximum viscosity as the polymer swells in water to form a gel. Prepare a second carbopol solution containing the MR contrast agent MnCl2 to a final concentration of 0.040 mM; this solution is referred to as the doped tracer fluid.</li>
	<li>Characterize the flow behavior, or rheology, of the carbopol solutions with a TA Instruments AR-G2 rheometer (New Castle, DE) with a standard Couette geometry (14 mm diam. x 42 mm height) at a fluid temperature of 25 &deg;C. For shear viscosity, use a steady state shear stress sweep from 0.1 500 Pa in logarithmic mode with 10 points/decade and 5% tolerance. Methods are described in [5].</li>
</ol>
In this work, the rheological properties of the two solutions were indistinguishable and are illustrated in Fig. 7, data were fit to a power law model and show shear thinning behavior.
Characterize the viscoelastic properties of the 0.2% w/w carbopol solution with small amplitude oscillatory testing. Perform the dynamic testing at a fixed stress of 1 Pa, which corresponds to the linear viscoelastic region. Measure strain over a frequency sweep from 628 - 0.63 rad/s (100-0.10 Hz) in logarithmic mode with 10 points/decade. 
The storage and loss moduli, G&rsquo; and G&rdquo; respectively, are shown in Fig. 8. The curves are characteristic of a gel system with G&rsquo;&gt;G&rdquo; and G&rsquo; fairly constant [5]. Values of tan(&delta;) = G&rdquo;/G&rsquo; increase from 0.05 at lower frequencies to 0.3 &minus; 0.5 at higher frequency. The corresponding phase lag (&delta;) followed the same trend, with the limits being &delta;=0 for Hookean solids and &delta;=&pi;/2 for Newtonian fluids. 
<ol start="3">
	<li>Evaluate the relative contribution of viscous forces to inertial forces during flow using the Reynolds number. Since the cross section of each plate varies, the average flow rate through the plate and the Reynolds number is calculated and given in Table 1. </li>
</ol>
These Reynolds number values are much less than 1.0 and characterize flows in which viscous forces dominate the inertial forces. In other words, the mixing is by laminar stretching and shearing rather than turbulence. 
4. MR data acquisition
<ol>
	<li>Select an appropriate radio frequency coil.</li>
</ol>
This work uses a solenoid with four turns, encasing a cylindrical volume 60 mm in diameter and 60 mm long. This coil closely fits the PVC pipe and achieved a good signal-to-noise ratio of the signal.
<ol start="2">
	<li>Run a multi-slice gradient echo sequence and acquire MR images. </li>
</ol>
This pulse sequence was chosen since the signal intensity is sensitive to material spin-lattice relaxation time. The relative signal intensity between two materials with different relaxation times is calculated from an equation. The signal intensity differences, total acquisition time for the image relative to the influence of molecular diffusion during the image acquisition all need to be considered in selecting the appropriate experimental parameters. Additionally, the concentration of the contrast agent (MnCl2) is chosen such that the signal intensity changes resulting from contrast agent concentration are linear. The addition of MnCl2 decreases the spin-lattice relaxation time (T1) of the test fluid from 2.998 s (undoped) to 0.515 s (doped). The doped Carbopol solution appears brighter than the undoped Carbopol solution in the images since the intensity is highly weighted by the spin lattice relaxation time. The pulse sequence parameters are an echo time (TE) of 2 ms and a repetition time (TR) of 30 ms; the field of view (FOV) is 64 mm per 128 encodings which yields an in-plane spatial resolution of 0.5 mm/voxel. With this multi-slice sequence, we acquire 32 cross sectional slices of thickness 1.4 mm per imaging slice.
5. Imaging the fluid
<ol>
	<li>Pump both the major and minor components through the mixer until steady flow is achieved. The relative flow rate of the major and minor components is 10:1. Simultaneously stop the pumps and image the fluid in the mixer. The MR sequence does not include flow compensation; to avoid motion artifacts, the imaging is performed on quiescent liquid. Imaging time is on the order of 1-4 minutes. </li>
	<li>Reposition the mixer several times to image cylindrical volumes at different axial locations.</li>
</ol>
In this study, several cylindrical volumes of the mixer are imaged and can be located in Fig. 9. The volume is chosen by sliding the mixer tube axially through the magnet, until the desired volume is in the sweet spot defined by the center of the NMR coil in the center of the magnet. 
<ol start="3">
	<li>Analyze the MR data with image analysis procedures to document the spatial distribution of component concentrations. The relationship between normalized signal intensity (x) and the fraction of doped fluid (y) in this study is y=1.419x-0.482 (R2=0.99). This relationship is relevant to analyze the mixing process.To illustrate the power of flow visualization using MRI, the following results are selected images at different axial locations.</li>
</ol>
6. Representative Results
Figure 10 illustrates images at the slit nozzle (injector) to show the cross sections as doped and undoped enter the first repeating unit. These images also clearly show the difference in signal intensity between 100% doped fluid and undoped fluid.
The SAR mixer effectively and uniformly splits flow as illustrated in the images of the H Platesdownstream from the 1st, 2nd, and 3rd mixing sections (Fig. 11, first row). The number of doped fluid &ldquo;stripes&rdquo; double through each mixing section. The second row of Fig. 11 illustrates the image analysis procedure that thresholds the images to &ldquo;1&rdquo;s (stripes) and &ldquo;0&rdquo;s (everything else). These processed images clearly illustrate the increase of interfacial area between the doped and undoped fluids as the fluid splits and recombines.
Sequential images through the second mixing section are shown in Fig. 12.
<img src="/files/ftp_upload/3493/3493table1.jpg" alt="Table 1"/> 
	Table 1. Cross sectional area of each plate and average velocity through the cross section, with corresponding Reynolds number (Re); defined for a power law fluid (PL), using an equivalent diameter.
<img src="/files/ftp_upload/3493/3493fig1.jpg" alt="Figure 1."/> 
	Figure 1. Photograph to illustrate flow through a split-and-recombine mixer using Carbopol dyed red as the minor component and undyed Carbopol solution as the major component.
<img src="/files/ftp_upload/3493/3493fig2.jpg" alt="Figure 2."/> 
	Figure 2. 2 Tesla super conducting magnet; for size reference, the conveyor is moving 3 avocados into the imaging region.
<img src="/files/ftp_upload/3493/3493fig3.jpg" alt="Figure 3."/> 
	Figure 3. Plate types and letter designations that are used to create a repeating unit in the SAR mixer.
<img src="/files/ftp_upload/3493/3493fig4.jpg" alt="Figure 4."/> 
	Figure 4. Schematic of the spilt-and-recombine mixer.
<img src="/files/ftp_upload/3493/3493fig5.jpg" alt="Figure 5."/> 
	Figure 5. 1 Tesla permanent-magnet-based imaging spectrometer (Aspect Imaging).
<img src="/files/ftp_upload/3493/3493fig6.jpg" alt="Figure 6."/> 
	Figure 6. Schematic of flow system.
<img src="/files/ftp_upload/3493/3493fig7.jpg" alt="Figure 7."/> 
	Figure 7. Apparent viscosity of the 0.2% Carbopol solution.
<img src="/files/ftp_upload/3493/3493fig8.jpg" alt="Figure 8."/> 
	Figure 8. Viscoelastic properties of the 0.2% Carbopol solution.
<img src="/files/ftp_upload/3493/3493fig9.jpg" alt="Figure 9."/> 
	Figure 9. Repeating units of the SAR mixer.
<img src="/files/ftp_upload/3493/3493fig10.jpg" alt="Figure 10."/> 
	Figure 10. Two images at the injector: the upstream section of the nozzle has a circular cross section for the doped fluid which gradually becomes a slit at the entrance to the first repeating unit of the SAR mixer.
<img src="/files/ftp_upload/3493/3493fig11.jpg" alt="Figure 11."/> 
	Figure 11. Fluid downstream of first, second, and third mixing sections, illustrated at Plate H.
<img src="/files/ftp_upload/3493/3493fig12.jpg" alt="Figure 12."/> 
	Figure 12. Sequences of 16 sequential images through the second mixing section. Please <a href="https://www.jove.com/files/ftp_upload/3493/3493fig12_lg.jpg">click here</a> to see a larger version of figure 12.

<ol>
	<li>Callaghan, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Nuclear Magnetic Resonance Microscopy. , (1991).</li><li>McCarthy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Magnetic Resonance Imaging in Foods. , (1994).</li><li>Sluijters, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixer.">Mixer.</a> US patent. , (1965).</li><li>van der Hoeven, J. C., Wimberger-Friedl, R., Meijer, H. E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneity+of+multilayers+produced+with+a+static+mixer.">Homogeneity of multilayers produced with a static mixer.</a> Polymer. Eng. Sci. 41, 32-42 (2001).</li><li>Steffe, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rheological Methods in Food Process Engineering. , (1996).</li><li>Lee, Y., McCarthy, M. J., McCarthy, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+of+mixing+in+a+two-component+batch+system+measured+using+MRI.">Extent of mixing in a two-component batch system measured using MRI.</a> J. Food. Eng. 50, 167-174 (2001).</li><li>McCarthy, K. L., Lee, Y., Green, J., McCarthy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+as+a+sensor+system+for+multiphase+mixing.">Magnetic resonance imaging as a sensor system for multiphase mixing.</a> Applied Magnetic Resonance. 22, 213-222 (2002).</li><li>Choi, Y. J., McCarthy, M. J., McCarthy, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+for+process+analysis:+Co-rotating+twin+screw+extruder.">MRI for process analysis: Co-rotating twin screw extruder.</a> Journal of Process Analytical Chemistry. 9, 72-84 (2004).</li><li>Rees, A. C., Davidson, J. F., Dennis, J. S., Fennell, P. S., Gladden, L. F., Hayhurst, A. N., Mantle, M. D., Muller, C. R., Sederman, 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=The+nature+of+the+flow+just+above+the+perforated+plate+distributor+of+a+gas-fluidised+bed,+as+imaged+using+magnetic+resonance.">The nature of the flow just above the perforated plate distributor of a gas-fluidised bed, as imaged using magnetic resonance.</a> Chemical Eng. Sci. 61, 6002-6015 (2006).</li><li>Stevenson, R., Harrison, S. T. L., Mantle, M. D., Sederman, A. J., Moraczewski, T. L., Johns, M. L. <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+partial+suspension+in+stirred+mixing+cells+using+both+MRI+and+ERT.">Analysis of partial suspension in stirred mixing cells using both MRI and ERT.</a> Chem. Eng. Sci. 65, 1385-1393 (2010).</li><li>Benson, M. J., Elkins, C. J., Mobley, P. D., Alley, M. T., Eaton, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+concentration+field+measurements+in+a+mixing+layer+using+magnetic+resonance+imaging.">Three-dimensional concentration field measurements in a mixing layer using magnetic resonance imaging.</a> Exp. Fluids. 49, 43-55 (2010).</li></ol>

Author M. McCarthy is a consultant to Aspect Imaging, Ltd. Authors L. Bacca and W. Hartt are employees of Procter &amp; Gamble, Co.

Magnetic resonance imaging is a rapid and quantitative method for analysis of fluid mixing. The measurement requires a few minutes to make and provides concentration of fluid as a function of position in the split-and-recombine mixer. This technique is suitable for application to a wide range of mixing problems and geometries [6-11]. Limitations to the technique are that a nonmagnetic mixer must be constructed and used in the MRI equipment, and at least one of the materials must provide a sufficient signal for data acquisition. A sufficient signal requires an NMR active nuclei with a sufficient number density. 
 MRI can also be used to quantify mixing of solids and liquids, two liquids with significantly different rheological properties as well as mixing in reacting systems. The mixing of solids into a liquid will yield different images than those of the SAR mixer. In the mixing of solids the solid component signal decays rapidly and is not imaged; hence, the signal is from the liquid only and the concentration of solid is derived from the signal loss compared to pure liquid signal. 
 MRI mixing images provide an excellent test of computational mixing experiments. The image data provides insight on the importance of fluid rheological properties and deviations from ideal conditions. In Fig. 12 the deviations from ideally uniform layers of fluid are evident. The images obtained thus provide detailed data sets suitable for direct comparison with numerical predictions of complex flows.

<table border="1">
<tbody>
 <tr>
 <td>Material or equipment </td>
 <td>Supplier</td>
 </tr>
 <tr>
 <td>Aspect 1T imaging spectrometer</td>
 <td>Aspect Imaging (Shoham, Israel)</td>
 </tr>
 <tr>
 <td>AR-G2 rheometer </td>
 <td>TA Instruments, (New Castle, DE)</td>
 </tr>
 <tr>
 <td>Seepex metering pumps</td>
 <td>Seepex GmbH, (Bottrop, Germany)</td>
 </tr>
 <tr>
 <td>Carbopol</td>
 <td>The Lubrizol corporation, (Wickliffe, OH)</td>
 </tr>
 <tr>
 <td>Manganese Chloride 1M Solution</td>
 <td>Fisher Scientific (Pittsburgh, PA)</td>
 </tr>
 </tbody>
</table>

quantifying mixing using magnetic resonance imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Watch this Scientific Journal Video about Quantifying Mixing using Magnetic Resonance Imaging at JoVE.com

Quantifying Mixing using Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

quantifying-mixing-using-magnetic-resonance-imaging

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Biology

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In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

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Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

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This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

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Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

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Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

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functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
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purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.