The authors gratefully acknowledge the National Science Foundation (DMR-1409034) for support of this project. We also acknowledge the support of the National Institute of Standards and Technology, U.S. Department of Commerce, in providing the USANS facilities used in this work, where these facilities are supported in part by the National Science Foundation under Agreement No. DMR-0944772. The SANS experiments of this research were completed at ORNL&#39;s High Flux Isotope Reactor, which was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy.

All handling of chemicals should be carried out with proper personal protective equipment and within a safety hood. All samples exposed to ionizing radiation should be handled under the supervision of the facilities radioactive control technicians. This protocol was performed by individuals who had completed appropriate radiation safety training.
1. Preparation of P3HT in d-ODCB Solutions
<ol>
	<li>Sample Acquisition
	<ol>
		<li>Purchase 1 g of high regioregularity (&#62;90%) P3HT in the molecular weight range 15 - 40 K.</li>
		<li>Purchase 5 g of high purity (&#62;90 atom% deuterated) d-4 1,2-ODCB.</li>
	</ol>
	</li>
	<li>Sample Preparation
	<ol>
		<li>Filter all d-ODCB solution with 0.45 &#181;m sieve into a glass vial.</li>
		<li>Combine 0.34 g of P3HT in 1.66 g of d-ODCB in a 5 g glass vial with foil lined cap. 
		NOTE: Throughout the sample creation and transfer process, minimize the ambient light intensity to which the sample is exposed at all times.</li>
		<li>Add a magnetic stir bar to the vial, secure the cap, and seal with parafilm.</li>
		<li>Wrap vial entirely in aluminum foil to prevent any light exposure to solution.</li>
		<li>Place sample on hot plate at 70 &#176;C for 1 - 3 h with the magnetic stir bar enabled.</li>
		<li>Remove from heat and stirring once solution is completely homogeneous (preferably leaving the sample heating/stirring overnight to ensure complete dispersion).</li>
		<li>Transfer solution from vial to a properly cleaned (with separate rinses of acetone and water) 1 or 2 mm thick quartz banjo cell using a glass pipette. 
		NOTE: Heating the glass pipette in a heating oven to 70 &#176;C immediately before transfer greatly simplifies this process.</li>
		<li>Affix banjo cell cap and seal with parafilm.</li>
		<li>Place banjo cell in complete darkness (i.e. inside a sealed box or wrapped in aluminum foil).</li>
		<li>In a similar fashion assemble a sample which contains only d-ODCB (filled to capacity) and an empty banjo cell, to act as the solvent background and empty cell, respectively, for the scattering experiments.</li>
	</ol>
	</li>
</ol>
2. Neutron Scattering Experiments
<ol>
	<li>SANS Experiments in the &#34;dark&#34; environment
	<ol>
		<li>With assistance from the&#160;instrument scientist, ensure a sample stage is affixed with the required temperature controls capable of directing a temperature ramp from 70 -&#160;20 &#176;C.</li>
		<li>Place the banjo cells in the appropriately sized holding blocks, secure, and label.</li>
		<li>Wrap entire block with 0.1 mm thick aluminum foil to ensure no ambient light is incident to the sample. Minimize deformations of the foil to ensure proper fitting of the wrapped block in the sample stage. Place this wrapped block and sample within the sample stage.</li>
		<li>With&#160;assistance from&#160;the instrument scientist, complete the proper instrument alignment and calibration using the appropriate standard measurements. Set the detector distance close to its maximum setting (for instance at 18 m) to ensure access to the lowest Q region (~0.001 &#197;-1), ultimately allowing a full Q range of roughly 0.001-0.1 &#197;-1. This will allow probing of length scales up to ~500 nm.</li>
		<li>With the assistance of the instrument scientist, collect count rates for the P3HT and solvent samples and perform calculations to determine the amount of scattering time required to achieve total detector counts per sample of roughly 500,000 to 1,000,000, ensuring good statistical quality in the data.</li>
		<li>With this information, create a sample script which will direct the 70-20 &#176;C temperature ramp and data collection process. Choose a range of discrete temperature points to best cover the entire range within the given time restraints, for instance every 2 &#176;C. For every point on the ramp make 3 separate entries in the script: a change to the desired temperature, a wait period (~15 minutes) to allow the system to thermally equilibrate before scattering is collected, and the scattering measurement itself conducted over the appropriate time duration to achieve the required detector counts</li>
		<li>Once the instrument and script are prepared, execute the script and begin the experiment. Be sure to collect data for the solvent and empty cell as well (without temperature ramp). Additionally, collect transmission data for each sample and a blocked beam measurement.</li>
	</ol>
	</li>
	<li>SANS Experiments in the &#34;light&#34; environment
	<ol>
		<li>Upon completion of the &#34;dark&#34; experiment, move the samples from the stage, place on a secure benchtop, and remove all aluminum foil while observing radiation safety protocols.</li>
		<li>Position an optical illuminator featuring a halogen light source near the sample stage such that the leads effectively illuminate the sample slot in the stage associated with the scattering collection position.</li>
		<li>Using a calibrated light meter, record the light intensity provided by the illuminator at maximum intensity at the position where the sample will sit. Intensity values will vary with illuminator and sample stage configuration, however, illumination intensity of at least 5,000 lux is desired.</li>
		<li>Once this illumination setup is properly assembled, return the samples to the stage, ensure the illuminator is properly lighting the active sample, heat again to 70 &#176;C, allow proper equilibration, and repeat&#160;the data collection procedure performed on the dark samples, with the optical illuminator providing uninterrupted direct light exposure thorough the entire duration of this step.</li>
	</ol>
	</li>
	<li>USANS Experiments
	<ol>
		<li>Prepare USANS samples in a similar fashion using quartz banjo cells and place in copper or titanium blocks within a temperature controlled sample stage.</li>
		<li>With the help of the instrument scientist, align and calibrate the instrument employing the requisite number of buffers at the given neutron wavelength to allow analysis of Q values from roughly 10-5 -&#160;10-3 &#197;-1, allowing&#160;length scales on the order of microns to be probed.</li>
		<li>Develop the experimental script in a similar fashion to the SANS experiments, allowing for thermal equilibration and data collection at each temperature studied previously.</li>
		<li>Replicate the SANS experiments again, execute the script once under &#34;dark&#34; conditions and again under &#34;&#34;light&#34; conditions.</li>
	</ol>
	</li>
</ol>
3. Data Reduction and Analysis
<ol>
	<li>SANS reduction and analysis
	<ol>
		<li>Using the respective reduction program,32 input the data files for scattering, background (solvent), empty cell, blocked beam, and transmission measurements to accomplish proper background subtraction and conversion of scattering data to absolute units of intensity in cm-1.</li>
		<li>With the data appropriately reduced, begin analysis by fitting the experimental scattering data to a model that is the linear addition of two fitting equations, one representing the nanofibril aggregates through the elliptical cylinder model,33 and another taking into account the free chains in solution through the polymer excluded volume model.34,35 The equation below describes this combination model approach: 
		<img alt="Equation 1" src="/files/ftp_upload/56163/56163eq1.jpg" /> 
		In this equation, &#981;P3HT describes the total volume fraction of P3HT in the solution, <img alt="Equation 2" src="/files/ftp_upload/56163/56163eq2.jpg" /> is the volume fraction of aggregated P3HT present and modeled as an elliptical cylinder, PPEV is the free chain excluded volume form factor for P3HT, PECM describes the elliptical cylinder form factor for the aggregates, and <img alt="Equation 3" src="/files/ftp_upload/56163/56163eq3.jpg" /> and <img alt="Equation 4" src="/files/ftp_upload/56163/56163eq4.jpg" /> are the scattering length density (SLD) contrast between P3HT aggregates and the solvent and between the free P3HT chains and the solvent, respectively. SLD values for all components of the system can be calculated with a knowledge of their chemical composition and mass density and the use of an SLD calculator available as part of most neutron analysis programs or online.36</li>
		<li>Upon proper fitting procedures using NCNR Igor fitting macros37 or the SASView fitting program, acquire values for the key structural parameters for the gelled system at all temperatures in the light and dark, allowing quantification of the morphological evolution occurring throughout this process as a function of temperature and light exposure. These structural parameters include the cross-sectional areas of the nanofibrils, free chain radius of gyration (Rg) and Porod exponent, and a qualitative assessment of the total amount of material present in the nanofibril phase.</li>
	</ol>
	</li>
	<li>USANS reduction and analysis
	<ol>
		<li>Using the respective reduction program, input the scattering data and background data for each buffer to merge the data into a single reduced curve in absolute intensity units of cm-1.</li>
		<li>Analyze the data using a Guinier-Porod power law model which allows quantitative assessment of the aggregate scattering patterns probed by the USANS length scale, and allows acquisition of the aggregate Rg values.38 Fit using this method through NCNR Igor fitting macros37 or the SASView fitting program to allow comparison of macro-aggregate Rg across all temperatures and illumination conditions.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

First, looking at the SANS data as a function of temperature, the increase in the Elliptical Cylinder Model scale factor indicates a marked increase in the amount of P3HT present in the nanofibril phase, which isconsistent with the progression of the gelation process. Simultaneously, the decrease in free chain Rg paired with an increase in Porod exponent reveals that the deteriorating thermodynamic conditions associated with temperature decrease are causing a chain collapse in the P3HT chains still present in ...

Conjugated polymers promise functional materials that can be utilized in a broad range of devices, such as organic light emitting diodes, organic semiconductors, chemical sensors, and organic photovoltaics.1,2,3,4,5,6 A crucial aspect of the performance in these devices is the ordering and packing of the conjugated polymer in the solid state in the active layer.7,8,9,10,11,12,13,14 This morphology is&#160;largely&#160;pre-determined by both the conformation of the polymer chain in solution as well as the structures that&#160;evolve as these solutions are cast unto a substrate and the solvent is removed. By studying the structures present throughout a typical sol-gel transition of a model optoelectronic polymer in a suitable solvent, these systems can be effectively modeled and a quantitative glimpse into the self-assembly that occurs during material deposition can be obtained.15,16,17,18,19,20
Specifically, we examine the conjugated polymer benchmark P3HT in the solvent deuterated ortho-dichlorobenzene (ODCB), a polymer-solvent system which has seen extensive use due to its suitability for a variety of organic electronic device fabrication techniques.23,24,25 In this given solvent environment, P3HT chains begin to aggregate upon an appropriate environmental stimuli, such as temperature decrease or loss of solvent quality. The exact mechanism for this assembly process is under investigation, with one of the leading proposed pathways believed to be a gradual process where individual P3HT molecules &#960;-stack to form lamellar nano-aggregates known as nanofibrils, which then themselves agglomerate to form larger micron scale macro-aggregates.24 Understanding these pathways and the resultant structures formed is&#160;key to properly predicting and influencing the formation of optimal device active layer morphologies.
Towards this ultimate goal of more precisely directing the formation of these active layer architectures, there exists a need to develop additional experimental and industrial methods to non-destructively alter conjugated polymer morphology in-situ. One relatively new methodology centers around the use of light exposure as an inexpensive means for altering polymer chain morphology, with both computational and experimental results pointing towards its feasibility.25,26,27 Recent work by our laboratory has indicated the existence of a light induced alteration of the conjugated polymer-solvent interaction in a dilute solution, leading to a notable change in polymer chain size upon illumination.30,31 Here, we present a protocol to continue this work by effectively monitoring the effects of exposing a much more concentrated conjugated polymer solution to direct light&#160;throughout a gelation process that is directed by a thermostat-controlled temperature ramp. We employ neutron scattering as it allows robust analysis of structural parameters of the polymer-solvent sol-gel system on length scales from angstroms to microns, an ability not possible through other more common rheological or spectroscopic instrumental methods.16,17,30,31 Thus, by comparing the properly analyzed small and ultra-small angle neutron data for the assembly of gels formed under illumination to identical data collected in complete darkness, structural differences brought on by illumination-driven effects can be comprehensively identified and quantified.

<table><tbody><tr><td>M(106) poly(3-hexylthiophene-2,5-diyl) (P3HT)</td><td>Ossila</td><td>104934-50-1</td><td>Conjugated polymer</td></tr><tr><td>deuterated 1,2 ortho-dichlorobenzene (ODCB)</td><td>Sigma Aldrich</td><td>AC321260050</td><td>solvent</td></tr></tbody></table>

monitoring the effects of illumination on the structure of conjugated polymer gels using neutron scattering

We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and absence of white light exposure. By instituting a controlled temperature ramp, the gelation of these materials can be precisely monitored as they proceed through this structural evolution, which effectively mirrors the conditions experienced during the solution deposition phase of organic electronic device fabrication. Using small angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) along with appropriate fitting protocols we quantify the evolution of select structural parameters throughout this process. Thorough analysis indicates that continued light exposure throughout the gelation process significantly alters the structure of the ultimately formed gel. Specifically, the aggregation process of poly(3-hexylthiophene-2,5-diyl) (P3HT) nano-scale aggregates is negatively affected by the presence of illumination, ultimately resulting in the retardation of growth in conjugated polymer microstructures and the formation of smaller scale macro-aggregate clusters.

Through SANS and USANS experiments, the gelation process of P3HT in d-ODCB was effectively monitored from the dispersed solution state at 70 &#176;C to a fully gelled state at 20 &#176;C. These experiments were conducted in both complete darkness and under white light illumination. Figure 1 displays some example SANS reduced data curves from these experiments, with an example curve fit shown in Figure 2. From this data, the struc...

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Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented.

We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and ...

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5.7K Views.  University of Tennessee, Knoxville. Een protocol voor de analyse van gelen gevormd uit de opto-elektronische geconjugeerd polymeer poly(3-hexylthiophene-2,5-diyl) (P3HT) met behulp van kleine en ultrakleine hoek neutronen verstrooiing in zowel de aanwezigheid en de afwezigheid van verlichting wordt gepresenteerd.

Video: Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

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 ...

Engineering

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials.

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Bioengineering

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam&#160;rapidly induces radiation damage of photosensitive&#160;cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (&#62; 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein&#160;crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps.

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding ...

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.

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 ...

Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

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