Name: measuring the time-evolution of nanoscale materials with stopped-flow and small-angle neutron scattering
Uploaded: 2021-08-06T14:00:00
Description: Watch this Scientific Journal Video about Measuring the Time-Evolution of Nanoscale Materials with Stopped-Flow and Small-Angle Neutron Scattering at JoVE.com

Access to the NG3 VSANS was provided by the Center for High-Resolution Neutron Scattering, a partnership between the National Institute of Standards and Technology and the National Science Foundation under Agreement NO. DMR-2010792. M.H.L.N acknowledges the funding provided by Mitacs Globalink (Canada). The identification of any commercial products or trade names is to foster understanding and does not imply endorsement or recommendation by the National Institute of Standards and Technology.

1. Set up and initiate the stopped-flow system.
<ol>
	<li>Turn on all pump power supplies and dynamic mixers using the power switch.</li>
	<li>Initiate all pumps and valves in the stopped-flow system control graphical user interface (GUI) by entering the device configuration path and using the commands bus=qmixbus.Bus(), bus.open(), bus.start(), pump=qmixpump.Pump(), pump.enable(), and valve=ViciMultiposSelector()&#160;</strong...

The current procedure describes the mixing device and the steps to perform stopped-flow TR-SANS measurements. The device and protocol are optimized for low-viscosity liquid samples where the time scales of interest are &#8776;1 s to 5 min. For time scales greater than 5 min, manually mixing the samples and loading them into standard scattering cells may be easier and desirable, especially for high-viscosity samples, gels, or pastes. Accessing time scales less than 1 s requires a different mixing apparatus, lower total vo...

Small-angle neutron scattering (SANS) provides a unique way to measure the sizes, shapes, interactions, and organization of various materials on length scales from &#8776;1 nm to &#8776;100 nm1,2,3. Recent instruments, including VSANS (very small-angle neutron scattering) instruments with focusing mirrors, push the limits toward measuring even larger length scales up to &#8776;1000 nm4,5. In general, the unique scattering contrast inherent to neutron scattering methods offers several advantages in measuring the time-evolution of nanoscale structures, such as the aggregation of components in pharmaceutical formulations6, crosslinking and gelation reactions in polymer systems7,8, in meso crystallization of membrane proteins9,10, degradation and unfolding of proteins11,12, and growth of silica-based materials13,14,15. The unique scattering contrast makes time-resolved SANS (TR-SANS) a useful complement to other stopped-flow-based measurements.
Stopped-flow mixing methods often are implemented in small-angle X-ray scattering (SAXS)16,17,18,19,20,21, fluorescence spectroscopy22,23,24,25,26, and light scattering27,28,29,30,31,32 experiments to study kinetic processes on the millisecond time scales. An important difference between SANS and SAXS is that neutron scattering is a nondestructive characterization technique, and as such, SANS can be used to measure the same sample for hours or even days without ionizing radiation damage to the sample, which can happen during higher-flux X-ray scattering experiments33. As repeated SANS measurements will not alter the chemical structure of the probe molecule or sample, the time-evolution can be studied without effects of photobleaching, for example, which can complicate kinetics measurements that rely on fluorescence23,24. Moreover, SANS can be used to measure highly concentrated and optically opaque samples that are often difficult to characterize with light-based techniques such as dynamic light scattering.
In addition to providing structural information on the nanoscale, SANS can be used to probe the local composition of these structures through the variation in neutron scattering length density contrast. The scattering length density (SLD) of different elements varies randomly across the periodic table and varies with different isotopes of the same element. A commonly exploited example is hydrogen (1H or H) and deuterium (2H or D), which have vastly different neutron scattering lengths. Therefore, hydrogen-rich materials, such as surfactants, lipids, proteins, RNA, DNA, and other polymers, can be distinguished from deuterated solvents using SANS without significantly changing the physical properties of the system. However, it is important to note that H/D exchange can affect the density, hydrogen-bonding, and phase transition temperatures in the sample. Nevertheless, the unique sensitivity of SANS to hydrogen-rich materials is especially useful in soft matter research where the samples of interest have lower scattering contrast and signal in X-ray-based techniques such as SAXS. Isotopic substitution also makes SANS a powerful tool for studying molecular exchange kinetics in hydrogen-rich materials by simply mixing H-labeled and D-labeled molecules. Isotopic substitution is particularly useful in systems where bulky fluorescent dyes are larger than the surfactant or lipid molecules of interest and can influence the exchange kinetics34,35.
Time-resolved SANS measurements are advantageous because the measured intensity is a function of time, length scale, and SLD contrast. As such, TR-SANS experiments can be designed to probe the time-dependent changes in the spatial distributions and the compositions of the samples. These unique advantages of SANS have led to important insights into kinetic processes in many soft material systems such as surfactants36,37,38, emulsions39,40,41, lipids34,42,43,44,45,46,47,48,49,50, and polymers51,52,53,54,55,56,57,58,59,60,61,62. Most TR-SANS studies have focused on time scales of minutes to hours. However, many kinetic processes of interest occur on the second time scale and are essential for understanding the underlying mechanisms. Capturing these early time points requires that the solutions be rapidly mixed and measured in situ, in which the mixing is synced with data collection during stopped-flow light scattering27,28,29,30,31,32, fluorescence22,23,24,25,26, and X-ray16,17,18,19,20,21 experiments. This work describes the use of a sample environment designed to rapidly mix multiple liquid samples and inject the mixture into a quartz glass cell for TR-SANS measurements. The mixing device is an adaptation of the recently developed capillary rheoSANS device63 and uses multiple syringe pumps and valves to control the sample mixing and to automate cell cleaning. By connecting syringe pumps to a series of flow selector valves, multiple inlet streams can be repeatedly mixed, measured, rinsed, and dried to facilitate TR-SANS measurements on the seconds time scale.
The current procedure assumes that the samples of interest have been identified and prepared. We focus on the in situ mixing setup and methods to collect TR-SANS data. Neutron scattering data were collected on the VSANS instrument at the NIST Center for Neutron Research (NCNR); however, the procedure should be applicable to other SANS instruments. Readers interested in implementing similar protocols on other SANS instruments should consult with the local instrument scientists to determine the optimal instrument configuration to maximize neutron flux at the desired length scale and time scale most relevant to the kinetic processes of interest. The data presented here were collected using the high flux &#39;white beam&#39; configuration on VSANS to maximize neutron counts at the loss of spatial resolution5. The detector carriages were positioned to cover a range of scattering vectors (q), 0.005 &#197;-1 &#60; q &#60; 0.5 &#197;-1, corresponding to length scales of &#8776;130 nm to &#8776;13 nm. The scattering vector is defined as q = 4&#960;/&#955; sin (&#952;/2) in which &#955; is the neutron wavelength, and &#952; is the scattering angle.
The stopped-flow mixing device used for the TR-SANS measurements consists of multiple pumps, rinsing syringes, sample syringes, flow selectors, as well as adynamic mixer, sample cell, and mixed sample container, as shown in Figure 1. All sealed fluid paths are located inside an air-conditioned enclosure, which includes the syringes, valves, connection tubing, dynamic mixer, and sample cells. A programmable thermoelectric air conditioner is used to control the enclosure temperature in the range from 10 &#176;C to 50 &#176;C within &#177;1 &#176;C. Note that some of the enclosure insulation was removed to show the working parts of the device. The main mixing device enclosure is positioned on a translational stage on the NG3 VSANS beam line at the NCNR. The enclosure position is adjusted using the translation stage to position the sample cell in the path of the neutron beam (yellow dashed line).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62873/62873fig1v2.jpg" /> 
Figure 1: An example setup for combining stopped-flow mixing and small-angle neutron scattering measurements at the VSANS beamline at the NIST Center for Neutron Research. The setup contains four syringe pumps, two syringes for solvent rinsing and two syringes for sample injection, four pump selector valves, two mixer selector valves, a dynamic mixer, a flow-through quartz cell, and a mixed sample container. Incident neutrons scatter off the mixed sample located inside the sample cell. An insulated enclosure with quartz windows and a thermoelectric air-conditioned unit is&#160;used to control the sample and all equipment at a constant temperature. The yellow dashed line shows the neutron beam path. Scale bar = 10 cm. <a href="https://www.jove.com/files/ftp_upload/62873/62873fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The device depicted in Figure 1 is configured with two sample syringes, two rinsing syringes, and one sample cell. Corresponding flow diagrams for the different steps of the protocol are illustrated in Figure 2. The desired volumes of the two different samples are injected into the mixer and the sample cell (Figure 2A). Once the sample cell is filled, the Inlet Switch Valve (ISV) and Outlet Switch Valve (OSV) are closed to isolate the sample cell from the dynamic mixer and to prevent sample back diffusion into the cell during TR-SANS data collection (Figure 2B). Before the dynamic mixer, the connection tubing varies in length from 10 cm to 1 m and does not affect the mixing delay time. However, tubing connections between the dynamic mixer and the sample cell will affect the mixing delay time and the required sample injection volume. Precut stainless steel tubing with 0.04 inch (1 mm) inner diameter and 100 mm length are used to connect the dynamic mixer, the Mixer Selector Valves (MSV1 and MSV2), and the ISV and OSV. Fluorinated tubing with 1 mm inner diameter and 115 mm length is used to connect the ISV and OSV (or the dynamic mixer outlet) to the sample cell. The total void volume that influences the mixing delay time includes the mixer void volume (0.15 mL), the tubing between the mixer outlet and the sample cell inlet (0.09 mL), and the sample cell volume (0.16 mL). In this example, the total void volume is 0.4 mL. The internal void volumes of valves are negligible compared to the tubing, mixer, and sample cell void volumes. For example, the employed low-pressure selector valves (0.75 mm bore diameter) contain approximate void volumes of 4 &#181;L, while the high-pressure selector valves and switch valves (0.25 mm bore diameter) contain approximate void volumes of 0.5 &#181;L.
After the TR-SANS measurement is complete, the sample is pushed out of the cell with solvent, and rinse solvent is repeatedly pumped through the cell to remove the residual sample and clean the sample cell (Figure 2C). Note that the rinse syringes are connected to larger solvent&#160;reservoirs (e.g., water and ethanol) via pump selector values to ensure that adequate solvent volumes are available to clean the sample cell between measurement runs. Solvent sources, sample sources, and mixed sample containers that contain flammable liquids are positioned in a separate enclosure with no electrical equipment to eliminate all possible ignition sources. In addition, vapor-locking bottle caps are used to minimize flammable vapors and solvent evaporation. Finally, the sample cell is dried with a nitrogen gas stream to remove the residual rinse solvent (Figure 2D). The inlet nitrogen gas pressure to the mixer selector valve is regulated to approximately 2 bar (0.2 MPa, gauge pressure) using a manual pressure regulator located on the nitrogen gas cylinder. Once the sample cell is sufficiently cleaned and dried, a newly mixed sample is injected into the sample cell for the next measurement cycle (repeating the mixing and injection illustrated in the flow diagram in Figure 2A).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62873/62873fig2v2.jpg" /> 
Figure 2: Example flow diagram using one sample cell, two samples mixing, and two rinse solvents for cleaning. (A) Mixing of sample A (blue) and sample B (red), and then flowing the mixed sample (purple)&#160;into the sample cell. (B) During data collection, the stopped-flow device state where the ISV and OSV switch valves are closed to isolate the sample cell and prevent back diffusion of the sample during data collection. (C) The cleaning steps where the sample cell is rinsed with rinse solvent from SS1 (green) after data collection. (D) Drying step where the sample cell is dried with nitrogen gas (orange). Abbreviations: PSV = pump selector valve; MSV = mixer selector valve; OSV = outlet switch valve; ISV = inlet switch valve; SS1 = solvent source 1; SSA = sample source A; N2 = nitrogen gas source. <a href="https://www.jove.com/files/ftp_upload/62873/62873fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 shows flow diagrams for a slightly different version in which the mixing setup is configured with two separate sample cells connected to the same switch valves (Figure 3A). While TR-SANS data are collected in Sample Cell 1, Sample Cell 2 is rinsed (Figure 3B) and dried (Figure 3C). When the data collection is complete for Sample Cell 1, the Inlet Switch Valve directs a newly mixed sample into Sample Cell 2 for data collection (Figure 3D). While TR-SANS data are collected in Sample Cell 2, Sample Cell 1 is rinsed and dried (Figure 3E). This alternating, parallel process between two sample cells minimizes the time between subsequent sample injections and maximizes the use of neutron beam time.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62873/62873fig3v2.jpg" /> 
Figure 3: Example flow diagram using a two-sample cells, two samples mixing, and two rinse solvents for cleaning. (A) Mixing sample A (blue) and sample B (red) and then flowing the mixed sample (purple) into sample cell 1. (B) The stopped-flow device state during data collection on sample cell 1 while sample cell 2 is rinsed with solvent from SS1 (green). (C) The stopped-flow device state during data collection on sample cell 1 while sample cell 2 is dried with nitrogen gas (orange). (D) Once data collection of sample cell 1 is complete, a new sample (purple) is immediately mixed and flowed into sample cell 2. (E) The stopped-flow device state during data collection on sample cell 2 while sample cell 1 is rinsed with solvent from SS1 (green). While one sample cell is being measured, the other sample cell is being cleaned and dried. The stopped-flow measurement process alternates between two sample cells to minimize the time between subsequent sample mixing injections. Abbreviations: PSV = pump selector valve; MSV = mixer selector valve; OSV = outlet switch valve; ISV = inlet switch valve; SS1 = solvent source 1; SSA = sample source A; N2 = nitrogen gas source. <a href="https://www.jove.com/files/ftp_upload/62873/62873fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A step-by-step protocol is described below for connecting the pumps and tubing lines, priming the system, rinsing and drying the sample cell, and injecting the mixed sample. Although the single-cell configuration is demonstrated for simplicity (Figure 2), the flexible modular setup, protocol, and scripts can be easily modified to implement more syringe pumps, valves, mixers, or sample cell configurations, such as the two-sample cell configuration shown in Figure 3. Representative raw neutron count rate data collected throughout mixing and cleaning injection cycles are shown in Figure 4, while lipid exchange kinetics measured at 3 different temperatures and the extracted normalized scattered intensity corresponding to the fraction of lipids exchanged are shown in Figure 5 and Figure 6, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynamic mixer</td>
			<td>Analytical Scientific Instruments</td>
			<td>462-0150A</td>
			<td>Magnetically coupled rotor, binary dynamic mixer assembly (ternary type available), 0.15 mL dead volume (larger dead volume available)</td>
		</tr>
		<tr>
			<td>Fluoropolymer tubing</td>
			<td>IDEX Health &#38; Science</td>
			<td>1507L</td>
			<td>PFA Tubing Natural 1/16 inch OD x 0.040 inch ID x 50 ft</td>
		</tr>
		<tr>
			<td>Fluoropolymer 1/4-28 flangeless fittings</td>
			<td>IDEX Health &#38; Science</td>
			<td>XP-245</td>
			<td>PFA flangeless fitting with ferrules, 1/4-28 threading, 1/16 inch OD tubing</td>
		</tr>
		<tr>
			<td>Glass syringes</td>
			<td>Hamilton Company</td>
			<td>81660</td>
			<td>Hamilton 1000 series syringes, 10 mL (81660), model 1010 C syr, 1/4&#34;-28 thread termination, other volumes available</td>
		</tr>
		<tr>
			<td>High-pressure flow selector valves</td>
			<td>Vici Valco</td>
			<td>C85X-1570EUTB</td>
			<td>Vici 10 position selector valves, 15000 psi max, 0.25 mm bore, 1/16 inch OD tubing, 10-32 coned threaded ports, USB universal actuator</td>
		</tr>
		<tr>
			<td>High-pressure switch valves</td>
			<td>Vici Valco</td>
			<td>C82X-1574EUHB</td>
			<td>Vici 4 port switch valves, 15000 psi max, 0.25 mm bore, 1/16 inch OD tubing, 10-32 coned threaded ports, USB universal actuator</td>
		</tr>
		<tr>
			<td>High-pressure syringes</td>
			<td>Cetoni</td>
			<td>A2019000358</td>
			<td>3 mL stainless steel syringe, 510 bar max, 21 mL/min flow rate max</td>
		</tr>
		<tr>
			<td>Low-pressure flow selector valves</td>
			<td>Vici Valco</td>
			<td>C25-3180EUHB</td>
			<td>Vici 10 position selector valves, max 250 psi liquid, 0.75 mm bore, 1/16 inch OD tubing, 1/4-28 threaded ports, USB universal actuator</td>
		</tr>
		<tr>
			<td>neMESYS high-pressure syringe pumps</td>
			<td>Cetoni</td>
			<td>A3921000103</td>
			<td>Max force 2600 N</td>
		</tr>
		<tr>
			<td>neMESYS mid-pressure syringe pumps</td>
			<td>Cetoni</td>
			<td>A3921000131</td>
			<td>Max force 1000 N</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Cetoni</td>
			<td>A3921000127</td>
			<td>Base 600, supplies power for up to 4 high pressure pumps</td>
		</tr>
		<tr>
			<td>Quartz flow-through sample cell</td>
			<td>Starna Scientific</td>
			<td>3-2.30-Q-1/TC</td>
			<td>Quartz micro flow cells, 2 mm path length (1 mm available), 2 mm by 2 mm by 30 mm internal dimension</td>
		</tr>
		<tr>
			<td>Quartz windows</td>
			<td>Technical Glass Products</td>
			<td>NA</td>
			<td>GE 124 Clear fused quartz ground and polished plates, 11.75 inch by 23.75 inch by 0.375 inch thick</td>
		</tr>
		<tr>
			<td>Stainless steel 10-32 coned compression fittings</td>
			<td>IDEX Health &#38; Science</td>
			<td>U-321X, U-320X</td>
			<td>316 stainless steel ferrule (U-321X) and nut (U-320X) -Valco type, 10-32 coned, for 1/16 inch OD stainless steel tubing</td>
		</tr>
		<tr>
			<td>Stainless steel tubing</td>
			<td>IDEX Health &#38; Science</td>
			<td>U-102</td>
			<td>Stainless Steel Tubing 1/16 inch OD x 0.020 inch ID, 10 cm, various precut lengths available</td>
		</tr>
		<tr>
			<td>Syringe pump control software</td>
			<td>Cetoni</td>
			<td>T6000000004</td>
			<td>QmixElements software for nemesys pumps, QmixSDK software development kit</td>
		</tr>
		<tr>
			<td>Thermoelectric air conditioner</td>
			<td>EIC Solutions</td>
			<td>AAC-140C-4XT-HC</td>
			<td>Thermoelectric air conditioner mounted on insulated enclosure to control the pump, valve, mixer, and sample temperature</td>
		</tr>
		<tr>
			<td>T-slot railing</td>
			<td>McMaster-Carr</td>
			<td>47065T103</td>
			<td>Aluminum t-slotted railing (1.5 inch by 1.5 inch) cut to various lengths</td>
		</tr>
		<tr>
			<td>Vapor locking bottle caps&#160;</td>
			<td>Cole-Parmer</td>
			<td>EW-12018-02</td>
			<td>Four 304 SS port inserts, 1/4&#34;-28 threads, GL45 bottle cap size, PTFE body, SS threads, PP collar</td>
		</tr>
	</tbody>
</table>

measuring the time-evolution of nanoscale materials with stopped-flow and small-angle neutron scattering

This paper presents the use of a stopped-flow small-angle neutron-scattering (SANS) sample environment to quickly mix liquid samples and study nanoscale kinetic processes on time scales of seconds to minutes. The stopped-flow sample environment uses commercially available syringe pumps to mix the desired volumes of liquid samples that are then injected through a dynamic mixer into a quartz glass cell in approximately 1 s. Time-resolved SANS data acquisition is synced with the sample mixing to follow the evolution of the nanostructure in solution after mixing. 
To make the most efficient use of neutron beam time, we use a series of flow selector valves to automatically load, rinse, and dry the cell between measurements, allowing for repeated data collection throughout multiple sample injections. Sample injections are repeated until sufficient neutron scattering statistics are collected. The mixing setup can be programmed to systematically vary conditions to measure the kinetics at different mixing ratios, sample concentrations, additive concentrations, and temperatures. The minimum sample volume required per injection is approximately 150 &#181;L depending on the path length of the quartz cell. 
Representative results using this stopped-flow sample environment are presented for rapid lipid exchange kinetics in the presence of an additive, cyclodextrin. The vesicles exchange outer-leaflet (exterior) lipids on the order of seconds and fully exchange both interior and exterior lipids within hours. Measuring lipid exchange kinetics requires in situ mixing to capture the faster (seconds) and slower (minutes) processes and extract the kinetic rate constants. The same sample environment can also be used to probe molecular exchange in other types of liquid samples such as lipid nanoparticles, proteins, surfactants, polymers, emulsions, or inorganic nanoparticles. Measuring the nanoscale structural transformations and kinetics of exchanging or reacting systems will provide new insights into processes that evolve at the nanoscale.

The representative neutron data shown here measure lipid exchange kinetics in the presence of methyl-&#946;-cyclodextrin (m&#946;CD), an additive that catalyzes the lipid exchange between vesicles with the exchange rate (ke)66,67. Previous fluorescence studies have shown that ke depends on the m&#946;CD concentration, and the half-life of the exchange process is on the order of minutes68. The present experiments use s...

Watch this Scientific Journal Video about Measuring the Time-Evolution of Nanoscale Materials with Stopped-Flow and Small-Angle Neutron Scattering at JoVE.com

Measuring the Time-Evolution of Nanoscale Materials with Stopped-Flow and Small-Angle Neutron Scattering

This protocol presents the use of a stopped-flow sample environment to quickly mix multiple liquid solutions in situ during a small-angle neutron scattering measurement and to study kinetic processes on nanometer length scales and second time scales.

This paper presents the use of a stopped-flow small-angle neutron-scattering (SANS) sample environment to quickly mix liquid samples and study nanoscale ...

Stop flow small angle scattering or stop flow SANS allows us to study how nanoscale materials evolve on set timescales of seconds to minutes. The contrast between hydrogen and deuterium unique to neutron scattering makes this technique particularly useful for studying materials rich in hydrogen such as lipids, proteins, and polymers. If you're interested in using stop flow stands to study your materials of interest, contact US local instrument scientist at a scattering facility to help plan your experiment.Demonstrating the procedure will be Ryan Murphy a chemical engineer at the National Institute of Standards and Technology, or NIST, Center for Neutron Research, or the NCNR and Brian Maranville a physicist also at the NCNR. To begin turn on all pump power supplies and dynamic mixers. Using the power switch initiate all pumps and valves in the stopped flow system controlled GUI by entering the device configuration path and commands mentioned in the text manuscript calibrate the pumps before attaching syringes.After ensuring the pumps have been calibrated. Screw in clean syringe barrels to the connection at the top of the pump. Ensure the syringe mount head is loosened before dispensing the fill volume so that the syringe does not break due to excessive force from the syringe piston.Then screw the syringe piston into the connection at the bottom of the pump. After the syringe barrel and syringe piston are connected to the pump, dispense the fill volume. After the piston stops moving tighten the syringe mount head.Then connect the tubing to the sample and solvent sources, syringes, valves, mixers, sample cells and mixed sample container. Define all tubing and valve connections made in the stopped flow system controlled GUI by typing in the corresponding port number connections made to each valve. To load the sample aspirate the desired sample and solvent volumes from their sources into the sample syringes through the pump selector valves.Then prime the system by dispensing all air from the syringes, tubing lines, and valves, ensuring that enough liquid volume is dispensed from each syringe. To remove all the air bubbles, once the air has been purged from the system, perform at least one sample injection by clicking the cell labeled Start mixing experiment in the controlled GUI, with this cell actively selected. Click on the run button at the top of the controlled GUI or press the shift and enter keys together on the keyboard.Visually inspect the sample cell for air bubbles. If bubbles are present repeat the purging steps, otherwise proceed to define the remaining experiment protocol steps. To define the stopped flow mixing protocol.Enter the temperature set point of the programmable air conditioner unit that controls the temperature of the insulated enclosures surrounding the stopped flow device. Then enter all rinsing sequence steps by typing in the appropriate volumes, flow rates, times and number of repetitions into the controlled GUI. Next define all sample injection sequence steps by typing the appropriate volumes, flow rates and times into the stopped flow system controlled GUI finally calculate the total time of a single stopped flow data collection cycle to define the SANS parameters.Set the total VSANS data acquisition time in the SANS instrument controlled GUI to the previously calculated cycle time. Then set the sample transmission measurement time to 100 seconds and turn on the event mode data collection. To measure the background scattering.Ensure that the local instrument shutter is closed before attaching the blocked beam sample to the back of the sample aperture. Then after securing the local instrument environment open the local instrument shutter. Next, define the blocked beam scattering data acquisition time in the software and collect blocked beam scattering data counting for the same duration as the longest scattering data acquisition time.Once the data collection is complete close the instrument shutter and remove the blocked beam sample from the sample aperture. Then proceed to measure the empty cell scattering before beginning the stopped flow experiment. Ensure that the local instrument area is secure.Then open the local instrument beam shutter. Begin SANS data collection using the SANS instrument control software on the instrument computer by dragging and dropping the desired runs into the instrument queue. Next, start the stopped flow mixing experiment in the controlled GUI and confirm that the defined stopped flow mixing protocol has started operating.Then add the transmission run to the bottom of the queue. The transmission will be collected after the scattering run finishes. After downloading the scattering data file and associated event files from the server bin the scattering data to the desired time bins by entering the appropriate commands as indicated in the text manuscript.Then reduce the bin scattering data using the software provided in the beam line to analyze the time resolved SANS data, calculate the process time of interest from the measurement times using the equation provided in the text manuscript. Then extract the kinetic parameters of interest from the change in Q dependent intensity as a function of process time. The measured neutron count rates of salt buffer background over multiple injection cycles consisting of nine rinsing steps.One drawing step and one sample injection step are shown here. The individual H labeled and D labeled lipid solutions were mixed at time T mix and immediately flowed into the sample cell. The measurement neutron count rate spiked and reached a maximum value when the sample cell was filled at T fill.The neutron counts from the mixed lipid vesicles sample at three different temperatures are plotted as a function of the corrected process timescale which is the process time of interest corrected for the steady state flow period and delay time. SANS data were collected continuously in event mode which can then be post processed into the desired time bins. The lipid exchange kinetics were measured at 36 degrees 30 degrees and 20 degrees Celsius.These data were bend into equal time bins of three seconds and are representative of the time and length scale dependent information that can be gained from a TR SANS measurement. The normalized intensity is plotted as a function of the process time for the different temperatures. Combining stop flow mixing and time resolve SANS provides new opportunities to explore structural evolutions in nano scan materials ranging from the lipid nanoparticles and next generation medicines and vaccines to geo polymers used in green building materials.

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Research

JoVE Journal

Engineering

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2 In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61BM system correspond well with literature data. The P3HT:PC61BM blend separates onto PC61BM-rich and P3HT-rich domains. Mechanical properties of PC61BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution.

Organic photovoltaic  (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale  inhomogeneity of OPV materials affects performance ...

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Using Neutron Spin Echo Resolved Grazing Incidence Scattering to Investigate Organic Solar Cell Materials

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped crystallites. Neutrons are passed through two well defined regions of magnetic field; one before and one after the sample. The two magnetic field regions have opposite polarity and are tuned such that neutrons travelling through both regions, without being perturbed, will undergo the same number of precessions in opposing directions. In this case the neutron precession in the second arm is said to &#34;echo&#34; the first, and the original polarization of the beam is preserved. If the neutron interacts with a sample and scatters elastically the path through the second arm is not the same as the first and the original polarization is not recovered. Depolarization of the neutron beam is a highly sensitive probe at very small angles (&#60;50&#160;&#956;rad) but still allows a high intensity, divergent beam to be used. The decrease in polarization of the beam reflected from the sample as compared to that from the reference sample can be directly related to structure within the sample.
In comparison to scattering observed in neutron reflection measurements the SERGIS signals are often weak and are unlikely to be observed if the in-plane structures within the sample under investigation are dilute, disordered, small in size and polydisperse or the neutron scattering contrast is low. Therefore, good results will most likely be obtained using the SERGIS technique if the sample being measured consist of thin films on a flat substrate and contain scattering features that contains a high density of moderately sized features (30 nm to 5 &#181;m) which scatter neutrons strongly or the features are arranged on a lattice. An advantage of the SERGIS technique is that it can probe structures in the plane of the sample.

Progress has been made in utilizing spin echo resolved grazing incidence scattering (SERGIS) as a neutron scattering technique to probe the length-scales in irregular samples. Crystallites of [6,6]-phenyl-C61-butyric acid methyl ester have been probed using the SERGIS technique and the results confirmed by optical and atomic force microscopy.

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped ...

In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries

Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2 However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3 This research is challenging, and we outline a method to address these challenges using in situ NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries.
We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the &#8216;roll-over&#8217; cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ NPD experiment and initial directions are presented on how to analyze such complex in situ data.

In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries

We describe the design and construction of an electrochemical cell for the examination of electrode materials using in situ neutron powder diffraction (NPD). We briefly comment on alternate in situ NPD cell designs and discuss methods for the analysis of the corresponding in situ NPD data produced using this cell.

Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Quantification of Hydrogen Concentrations in Surface and Interface Layers and Bulk Materials through Depth Profiling with Nuclear Reaction Analysis

Nuclear reaction analysis (NRA) via the resonant 1H(15N,&#945;&#947;)12C reaction is a highly effective method of depth profiling that quantitatively and non-destructively reveals the hydrogen density distribution at surfaces, at interfaces, and in the volume of solid materials with high depth resolution. The technique applies a 15N ion beam of 6.385 MeV provided by an electrostatic accelerator and specifically detects the 1H isotope in depths up to about 2 &#956;m from the target surface. Surface H coverages are measured with a sensitivity in the order of ~1013 cm-2 (~1% of a typical atomic monolayer density) and H volume concentrations with a detection limit of ~1018 cm-3 (~100 at. ppm). The near-surface depth resolution is 2-5 nm for surface-normal 15N ion incidence onto the target and can be enhanced to values below 1 nm for very flat targets by adopting a surface-grazing incidence geometry. The method is versatile and readily applied to any high vacuum compatible homogeneous material with a smooth surface (no pores). Electrically conductive targets usually tolerate the ion beam irradiation with negligible degradation. Hydrogen quantitation and correct depth analysis require knowledge of the elementary composition (besides hydrogen) and mass density of the target material. Especially in combination with ultra-high vacuum methods for in-situ target preparation and characterization, 1H(15N,&#945;&#947;)12C NRA is ideally suited for hydrogen analysis at atomically controlled surfaces and nanostructured interfaces. We exemplarily demonstrate here the application of 15N NRA at the MALT Tandem accelerator facility of the University of Tokyo to (1) quantitatively measure the surface coverage and the bulk concentration of hydrogen in the near-surface region of a H2 exposed Pd(110) single crystal, and (2) to determine the depth location and layer density of hydrogen near the interfaces of thin SiO2 films on Si(100).

We illustrate the application of 1H(15N,&#945;&#947;)12C resonant nuclear reaction analysis (NRA) to quantitatively evaluate the density of hydrogen atoms on the surface, in the volume, and at an interfacial layer of solid materials. The near-surface hydrogen depth profiling of a Pd(110) single crystal and of SiO2/Si(100) stacks is described.

Nuclear reaction analysis (NRA) via the resonant 1H(15N,&#945;&#947;)12C reaction is a highly effective method of depth profiling that quantitatively and ...

Dielectric RheoSANS &#8212; Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids

A procedure for the operation of a new dielectric RheoSANS instrument capable of simultaneous interrogation of the electrical, mechanical and microstructural properties of complex fluids is presented. The instrument consists of a Couette geometry contained within a modified forced convection oven mounted on a commercial rheometer. This instrument is available for use on the small angle neutron scattering (SANS) beamlines at the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR). The Couette geometry is machined to be transparent to neutrons and provides for measurement of the electrical properties and microstructural properties of a sample confined between titanium cylinders while the sample undergoes arbitrary deformation. Synchronization of these measurements is enabled through the use of a customizable program that monitors and controls the execution of predetermined experimental protocols. Described here is a protocol to perform a flow sweep experiment where the shear rate is logarithmically stepped from a maximum value to a minimum value holding at each step for a specified period of time while frequency dependent dielectric measurements are made. Representative results are shown from a sample consisting of a gel composed of carbon black aggregates dispersed in propylene carbonate. As the gel undergoes steady shear, the carbon black network is mechanically deformed, which causes an initial decrease in conductivity associated with the breaking of bonds comprising the carbon black network. However, at higher shear rates, the conductivity recovers associated with the onset of shear thickening. Overall, these results demonstrate the utility of the simultaneous measurement of the rheo-electro-microstructural properties of these suspensions using the dielectric RheoSANS geometry.

Dielectric RheoSANS &amp;#8212; Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids

Here, we present a procedure for the measurement of simultaneous impedance, rheology and neutron scattering from soft matter materials under shear flow.

A procedure for the operation of a new dielectric RheoSANS instrument capable of simultaneous interrogation of the electrical, mechanical and ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

A Novel Method for In Situ Electromechanical Characterization of Nanoscale Specimens

Electrically assisted deformation (EAD) is increasingly being used to improve the formability of metals during processes such as sheet metal rolling and forging. Adoption of this technique is proceeding despite disagreement concerning the underlying mechanism responsible for EAD. The experimental procedure described herein enables a more explicit study compared to previous EAD research by removing thermal effects, which are responsible for disagreement in interpreting previous EAD results. Furthermore, as the procedure described here enables EAD observation in situ and in real time in a transmission electron microscope (TEM), it is superior to existing post-mortem methods that observe EAD effects post-test. Test samples consist of a single crystal copper (SCC) foil having a free-standing tensile test section of nanoscale thickness, fabricated using a combination of laser and ion beam milling. The SCC is mounted to an etched silicon base that provides mechanical support and electrical isolation while serving as a heat sink. Using this geometry, even at high current density (~3,500 A/mm2), the test section experiences a negligible temperature increase (&#60;0.02 &#176;C), thus eliminating Joule heating effects. Monitoring material deformation and identifying the corresponding changes to microstructures, e.g. dislocations, are accomplished by acquiring and analyzing a series of TEM images. Our sample preparation and in situ experiment procedures are robust and versatile as they can be readily utilized to test materials with different microstructures, e.g., single and polycrystalline copper.

A Novel Method for In Situ Electromechanical Characterization of Nanoscale Specimens

Isolating electrical and thermal effects on electrically assisted deformation (EAD) is very difficult using macroscopic samples. Metallic sample micro- and nanostructures together with a custom test procedure have been developed to evaluate the impact of applied current on the formation without joule heating and evolution of dislocations on these samples.

Electrically assisted deformation (EAD) is increasingly being used to improve the formability of metals during processes such as sheet metal rolling and ...