We acknowledge Dr. Dietmar Schwahn (Forschungszentrum J&#252;lich GmbH) for support and stimulating discussions regarding the upgrades performed on the KWS-2 in 2010-2015. The constant help from the Central Institute of Engineering, Electronics, and Analytics (ZEA) and the JCNS-1 (Neutron Scattering) and JCNS-2 (Scattering Methods) Institutes in Forschungszentrum J&#252;lich GmbH during the design, installation, and commission of components, devices, and control software for the new working modes of the KWS-2 is gratefully acknowledged. We are thankful to Matthew Binns and Christopher J. Van Leeuwen (both at Louisiana State University) for the professional editing of this manuscript.

1. Loading the Sample Cells
<ol>
	<li>Disperse polystyrene spherical particles with different sizes (radii of R = 150, 350, 500, 1,000, and 4,000 &#197;) and a size polydispersity of &#963;R <img alt="Equation 5" src="/files/ftp_upload/54639/54639eq5.jpg" /> 8% in a water solution (a mixture of 90% D2O and 10% H2O) at a volume fraction of 1%.</li>
	<li>Transfer the six solutions of polystyrene particles in D2O/H2O, the solution of C28H57-PEO5 in D2O, and the D2O/H2O and D2O solvents to quartz cells (Figure 3) using Pasteur pipettes. Fill each of the quartz cells up to the neck. Close the quartz cells with their stoppers. 
	NOTE: The operation of filling the quartz cells with samples must be carried out in the sample preparation lab of FRM II by complying with the special working conditions defined there.</li>
	<li>Install the filled quartz cells into the Al-cartridges of the sample holder (Figures 3). Place each filled quartz cell in a cavity of the cartridge and check whether the quartz cell is filled with enough sample by determining whether the sample entirely covers the window opening in the cartridge that is provided for neutrons. Place the correction and the standard samples (the empty quartz cell, the boron-carbide plate, and the Plexiglas plate) in additional locations on the cartridge and leave one location free for the empty beam measurement.</li>
	<li>Cover the cartridge with the Cd-coated Al-cover plates (Figure 3) using cap screws (M3x5). Fix the cartridge on the Al-frame of the sample holder by using the special Al screws (Figure 4).</li>
</ol>
2. Positioning the Sample Holder/Sample Environment on the Sample Stage
<ol>
	<li>Determine the necessary space at the sample position of the sample holder by adjusting the length of the collimation nose (Figure 5). Select the appropriate configuration for the sample holder from the stored configurations in the collimation nose control system.</li>
	<li>Install the appropriate sample holder/sample environment on the sample stage in the predetermined position on the optical breadboard using Allen screws, M6x40 (Figure 6).</li>
	<li>Close the motorized sliding lead door (Figure 5) by using the knobs on its outer side, keeping the control knobs activated until the door reaches the end position, which will be indicated by a luminous signal. 
	Caution: Without the door completely closed and the end-switch activated, the beam shutter cannot be opened, either manually or from the measurement control software; the measurement software will require an additional check for this issue before starting the measurement.</li>
</ol>
3. Planning the Experiments
<ol>
	<li>Choose the adequate experimental configuration and mode for performing the investigation in a Q range that is appropriate for the length scale of the structures and the correlation effects revealed by the sample. Check the dynamic range of the instrument4 (Figure 7 and Table 2).</li>
	<li>Choose the adequate experimental configuration and mode for enabling proper intensity on the sample based on the estimated level of the scattered intensity by knowing the approximate size, concentration, and contrast factor that characterize the samples to be investigated8. This is done in order to optimize the measurement time for a targeted measured statistic and to match the sample stability in case of samples with a short stability time. Check the intensity map for different experimental configurations4 (Supplementary Figure 1 and Table 2).</li>
	<li>Choose the adequate experimental configuration and mode for providing a proper resolution based on an approximate knowledge of the degree of polydispersity in size and the concentration of the scattering objects in the sample8. This is done in order to enable the resolution of fine scattering features that occur due to ordering effects in the sample. Check for the possibility of tuning the wavelength resolution5 (Table 2).</li>
</ol>
4. Preparing the Measurement Software and Conducting and Visualizing the Experiment
<ol>
	<li>Start the measurement software by typing the KWS2TC in a terminal window at the measurement control computer of the KWS-2 instrument in order to activate the main menu (Supplementary Figure 3). Use the left set of functions to define the elementary motor positions (Configuration), to select samples and setup conditions (Definition), to start the measurement and supervise all motors (Control), and to supervise the actual detector (Live-Display).</li>
	<li>Select the Configuration function in the main menu in order to activate the kws2-Configuration menu (Supplementary Figure 4) to define the user data and configure the elementary motor positions and the set-points for the devices and fields on the sample.
	<ol>
		<li>Select the UserData function (Supplementary Figure 4) and fill the fields User name, E-mail, First part of filename, and Measurement comment in the User data menu (view/edit). Leave the menu by clicking Save. 
		Caution: Do not use special characters, like @, $, %, etc., for the file name prefix. Avoid using special characters throughout the entire experimental procedure.</li>
		<li>Select the Sample function (Supplementary Figure 4) in order to activate the Sample configuration menu (Supplementary Figure 4). Fill the fields Sample title, Sample beam window - Size, Sample thickness, and Comment for each sample and position selected from the left vertical list of the menu. Save each sample configuration after completing the definition. Leave the menu by clicking Close.</li>
		<li>Save all configurations under the File function at the upper set of functions in the Configuration menu.</li>
	</ol>
	</li>
	<li>Select the Definition function in the main menu (Supplementary Figure 3) in order to activate the kws2 Definition menu (Supplementary Figure 5) to define the experimental setup and the measurement program.
	<ol>
		<li>Select the Sample function in order to activate the Select Samples menu (Supplementary Figure 5).
		<ol>
			<li>Choose the twelve samples that must be measured (as shown in Figure 3) from the list of known samples in the left vertical field and move them to the Selected Samples field using the blue arrow. Order the list of the selected samples by using the blue vertical arrows.</li>
			<li>Check the sample parameters and adjust the name, thickness, and comment, if necessary. Leave the menu by clicking Save or Close.</li>
		</ol>
		</li>
		<li>Select the Detector function to activate the Definition of Measurements menu (Supplementary Figure 6).
		<ol>
			<li>Choose the static measurement type by selecting Standard in the Measurement area. In the End Conditions area, select the proper time unit for the field measuring time.</li>
			<li>In the Select Detector and Collimation Distances area, select the experimental setup and working mode by choosing the appropriate values for the wavelength (Selector field), detection distance (Detector Distance field), data acquisition mode (TOF field), aimed wavelength resolution (DLambda/Lambda field), measurement time (Time field), lenses configuration (Lenses field), and collimation distance (Collimation Distance field).</li>
			<li>Click the New button after one configuration is entirely defined in order to fix it and store it in the lower table. Define the next configuration and store it in a similar way until the entire set of configurations (Supplementary Figure 6) is completed. Hit Save or Close in the Definition of Measurements menu when the adjustment of the experimental setup and working modes is finished.</li>
			<li>Sort the list of measurements according to the three loops (sorting conditions) shown at the bottom of the menu that is generated by the program (Supplementary Figure 7). Remove the measurements that are not desired by marking the corresponding line and clicking the &#34;X&#34; button marked in red. Adjust the measurement time for each measurement as desired by checking the total defined measurement time in the central field, marked in red, in the kws2 Definition menu, which always remains active on the screen.</li>
			<li>Leave the menu with Save or Close and return to the kws2 Definition menu (Supplementary Figure 5). Close the kws2 Definition menu and return to the main menu (Supplementary Figure 3).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select the Control function in the main menu of the KWS2 measurement software (Supplementary Figure 3) in order to activate the Measurement control menu (Supplementary Figure 8).
	<ol>
		<li>Log in with the username and password that will be communicated by the instrument scientist and lock the session for generating the script, which will execute the commands of the uploaded measurement program. Select Loop Definition in order to check the uploaded measurement program.</li>
		<li>Press the Start button and answer the questions generated by the program about the actual status of the sample position door and the beam-shutter. The measurement program will be started. Select Current Values in order to enable the visualization of the ongoing measurement (the positions of motors and the status of instrument components, count rate, and evolution of the integral intensity in time). 
		NOTE: The integral intensity on the detector and the count rates of the detector and monitors are shown and can be used to change the measurement program, either individually or the entirety of the set of measurements.</li>
	</ol>
	</li>
	<li>Allow the measurements be conducted and completed according to the defined measurement program. 
	NOTE: The measurements can be interrupted or stopped by activating the Stop function and choosing the desired option (to continue, to stop the current measurement with or without saving the file, or to stop the entire program) when the integral intensity collected up to a certain time point is considered sufficient or when failures are identified in the measurements sequence.
	<ol>
		<li>Generate the Logbook of the measurement session by clicking on the Print button under the Loop Definitions option (Supplementary Figure 8) when the defined measurement program has been stopped or completed.</li>
	</ol>
	</li>
	<li>Select Live-Display in the main menu of the KWS2 measurement software (Supplementary Figure 3) in order to activate the KWSlive_MainWindow interface (Supplementary Figure 9).
	<ol>
		<li>At the Type screen, choose either GEDET or PSD in order to visualize data collected with the main or with the secondary (high-resolution) detector, respectively. Choose the visualization mode at Display Mode by selecting the three-dimensional (Surface), the two-dimensional (Contour), or the one-dimensional (Radial average) mode. Enter the plot options (linear or logarithmic scale) and the parameter values (wavelength and detection distance LD) in the fields of the Radial average options menu in order to enable the presentation of data as n intensity (uncorrected) versus Q.</li>
		<li>Choose a desired TOF-channel in order to visualize the data collected in TOF mode (with either the main or the secondary detector).</li>
	</ol>
	</li>
</ol>
5. Data Analysis
<ol>
	<li>Start the data processing software by typing the qtiKWS command in a terminal window of the data analysis computer of the KWS-2 instrument. Choose the New Script option on the right side of the main interface.</li>
	<li>Choose the DAN option in the upper functions menu of the main interface (Supplementary Figure 10) in order to activate the data analysis functions. Select the KWS-2 instrument from Options on the right-side menu in order to activate the data analysis mode for data measured on the KWS-2 using the main detector. Define the folder where the measured data files are located and where the corrected data files will be stored.</li>
	<li>Choose the Tools option and activate the Header(s) function on the right-side menu (Supplementary Figure 11) and generate the info-table that contains the files to be processed. Define the table name by clicking the left set of green arrows on the Header(s) field. Load the measured files by clicking the right set of green arrows on the Header(s) field and selecting the measured files. 
	NOTE: The info-table that contains the complete information about each measurement file is stored in the explorer-like menu in the lower part of the project. All results that will be generated later in the project will be stored there.</li>
	<li>Activate the Mask function in the right-side menu (Supplementary Figure 12) and generate the active mask that defines the area of the detector that will be considered for data processing. Enter values in the Edge and Beam-Stop fields for defining the lower-left and upper-right corners of the rectangular mask in the case of the analysis of isotropic scattering patterns.</li>
	<li>Activate the Sensitivity function in the right-side menu (Supplementary Figure 13) and generate the detector sensitivity for a certain configuration by entering in the marked green fields the run numbers for the measurements of the standard sample (Plexiglas), empty beam (EB), and blocked beam (B4C).
	<ol>
		<li>Click on the set of green arrows next to the yellow field (Transmission) for calculating the transmission of the standard sample. Generate and name the sensitivity matrix by selecting Calculate as New, and visualize the generated matrix by using the appropriate plot functions in the lower menu. Repeat this procedure in case of other configurations.</li>
	</ol>
	</li>
	<li>Activate the Data Processing function in the right-side menu (Supplementary Figure 14) and generate the correction and calibration table and the script table in order to correct, calibrate, and perform the radial averaging of the data.
	<ol>
		<li>Define the number of conditions used in the experiment by using the horizontal slider on the top of the right-side menu (red arrow). Fill the fields indicated with the yellow pencil by entering for each experimental condition the run numbers for the empty cell (EC), blocked beam (B4C), and standard samples-the Plexiglas (Abs. Cal. FS), empty beam for the standard correction (Abs. Cal. EB), and blocked beam for the standard correction (Abs. Cal. B4C).</li>
		<li>Enter the run numbers of measurements with strong forward scattering in the Center field. Enter the run number of an empty beam in the EB field and select the corresponding experimental condition for the calculation of the transmission of samples by checking the box next to the Tr (Ec-to-EB) function.</li>
		<li>Click each button indicated by a set of rotating green arrows on the vertical series of options (Supplementary Figure 14) in order to load the information needed for data processing from the defined files and to calculate the transmission of the empty cell. Click the head of each yellow column in order to define the column name.</li>
		<li>Click the New button in order to generate and name the table of the data files that will be processed. Click the Add button in order to load the data files that will be processed. Click the Tr button indicated with the set of rotating green arrows under the Script-Table Tools area in order to calculate the transmission of each sample. Check the results in the generated table (Supplementary Figure 14).</li>
		<li>Choose Project in the lower-right corner of the interface (Supplementary Figure 14) in order to save all results as tables or matrices in the current qtiKWS session (project). Click the I[x,y] button in order to perform the correction and calibration of two-dimensional data. Click the I(Q) button in order to perform the correction, calibration, and radial averaging of the data. Plot the results using the graphical functions under the Graph (Supplementary Figure 15) option. 
		NOTE: All results will be generated as external files that will be saved in the external folder that was defined in step 5.2, when, in the lower-right corner of the interface, File is chosen instead of Project.</li>
	</ol>
	</li>
	<li>Chose Tools in the right-side menu (Supplementary Figure 11) and activate the TOF|RT option (Supplementary Figure 16) in order to split the data that were collected with the main detector in the TOF working mode into single files corresponding to each time channel.
	<ol>
		<li>Click the TOF :: Calculate Parameters function and load one file, from which information about the TOF conditions is extracted. Click on the TOF|RT :: Sum vs Number :: Read function and load the file of interest, measured in real-time or TOF modes, in order to generate a sum-tof-file table like that shown on the left side of the working interface. Plot the integral intensity as a function of time channels (Supplementary Figure 16) from the sum-tof-file using the graphical options under the Graph function in the upper functions menu.</li>
		<li>Define the processing parameters in the fields of the TOF function. Click the TOF|RT :: All Selected Steps :: Proceed button in order to load the data files that will be separated into single files corresponding to each of the defined time slots. 
		NOTE: Files that contains data measured in every time slot are generated and stored in the file location defined in step 5.2 and receive the name of the original TOF file, followed by the number of the time slot.</li>
		<li>Proceed as in step 5.6 in order to analyze the data measured with improved resolution, corresponding to &#916;&#955;\&#955;aimed, using the chopper.</li>
	</ol>
	</li>
	<li>Select the KWS2-HRD instrument from Options on the right-side menu of the main interface (Supplementary Figure 17) in order to activate the data analysis mode for data measured with the KWS-2 using the secondary high-resolution detector. Activate the Mask function in the right-side menu (Supplementary Figure 18A) and generate the active mask that defines the active area of the detector.
	<ol>
		<li>Choose the DANP option in the upper functions menu of the main interface (Supplementary Figure 18B). Select the ASCII.2D option in the right-side menu. Activate the 2D Masking function in order to define a special sector on the detector that will be considered for the data analysis.</li>
		<li>Introduce the beam-stop center in the field Center. Select the mask matrix and the value 0 in Mask :: Conditions to neglect the outside area of the special mask. Choose the angular sector and click the colored button on the right side of the sector fields. Proceed with the data measured with the high resolution detector, as in steps 5.5 and 5.6.</li>
	</ol>
	</li>
	<li>Save the qtiKWS project (save functions under the File option of the upper menu).</li>
</ol>

<ol>
	<li>Gl&#228;ser, W., Petry, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+neutron+source+FRM+II.">The new neutron source FRM II.</a> Physica. B. 276-278, 30-32 (2000).</li><li>Radulescu, A., Pipich, V., Ioffe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assessment+of+neutron+delivery+system+for+small-angle+neutron+scattering+diffractometers+of+the+J&#252;lich+Centre+for+Neutron+Science+at+FRM+II.">Quality assessment of neutron delivery system for small-angle neutron scattering diffractometers of the J&#252;lich Centre for Neutron Science at FRM II.</a> Nucl. Instrum. Methods Phys. Res. Sect. A. 689, 1-6 (2012).</li><li>Radulescu, A., Ioffe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutron+guide+system+for+small-angle+neutron+scattering+instruments+of+the+J&#252;lich+Centre+for+Neutron+Science+at+FRM-II.">Neutron guide system for small-angle neutron scattering instruments of the J&#252;lich Centre for Neutron Science at FRM-II.</a> Nucl. Instrum. Methods Phys. Res. Sect. A. 586, 55-58 (2008).</li><li>Radulescu, A., Pipich, V., Frielinghaus, H., Appavou, M. S. K. W. S. -. 2. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=the+high+intensity/wide+Q-range+small+angle+neutron+diffractometer+for+soft-matter+and+biology+at+FRM+II.">the high intensity/wide Q-range small angle neutron diffractometer for soft-matter and biology at FRM II.</a> J. Phys.: Conf. Ser. 351, 012026 (2012).</li><li>Radulescu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+instrument+resolution+using+chopper+and+time+of+flight+at+the+small-angle+neutron+scattering+diffractometer+KWS-2.">Tuning the instrument resolution using chopper and time of flight at the small-angle neutron scattering diffractometer KWS-2.</a> J.Appl.Cryst. 48, 1849-1859 (2015).</li><li>Frielinghaus, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspherical+refractive+lenses+for+small-angle+neutron+scattering.">Aspherical refractive lenses for small-angle neutron scattering.</a> J. Appl. Cryst. 42, 681-690 (2009).</li><li>Radulescu, A., Fetters, L. J., Richter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+semicrystalline+polymer+morphologies+by+imaging-SANS.">Structural characterization of semicrystalline polymer morphologies by imaging-SANS.</a> J.Phys.: Conf.Ser. 340, 012089 (2012).</li><li>Zemb, T., Lindner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutron, X-rays and Light Scattering Methods Applied to Soft Condensed Matter. , (2002).</li><li>Pedersen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+small-angle+scattering+data+from+colloids+and+polymer+solutions:+modeling+and+least+square+fittings.">Analysis of small-angle scattering data from colloids and polymer solutions: modeling and least square fittings.</a> Adv. Colloid Interface Sci. 70, 171-210 (1997).</li><li>Barker, J. G., Pedersen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instrumental+Smearing+Effects+in+Radially+Symmetric+Small-Angle+Neutron+Scattering+by+Numerical+and+Analytical+Methods.">Instrumental Smearing Effects in Radially Symmetric Small-Angle Neutron Scattering by Numerical and Analytical Methods.</a> J. Appl. Cryst. 28, 105-114 (1995).</li><li>Hammouda, B., Mildner, D. F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+neutron+scattering+resolution+with+refractive+optics.">Small-angle neutron scattering resolution with refractive optics.</a> J. Appl. Cryst. 40, 250-259 (2007).</li><li>Vad, T., Sager, W. F. C., Zhang, J., Buitenhuis, J., Radulescu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+determination+of+resolution+function+parameters+from+small-angle+neutron+scattering+data+of+a+colloidal+SiO2+dispersion.">Experimental determination of resolution function parameters from small-angle neutron scattering data of a colloidal SiO2 dispersion.</a> J. Appl. Cryst. 43, 686-692 (2010).</li><li>Amann, M., Willner, L., Stellbrink, J., Radulescu, A., Richter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+concentration+dependence+of+the+aggregation+number+of+a+micellar+model+system+by+SANS.">Studying the concentration dependence of the aggregation number of a micellar model system by SANS.</a> Soft Matter. 11, 4208-4217 (2015).</li><li>Str&#246;bl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diploma Thesis. , (2008).</li><li>Dahdal, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Angle+Neutron+Scattering+Studies+of+Mineralization+on+BSA+Coated+Citrate+Capped+Gold+Nanoparticles+Used+as+a+Model+Surface+for+Membrane+Scaling+in+RO+Wastewater+Desalination.">Small-Angle Neutron Scattering Studies of Mineralization on BSA Coated Citrate Capped Gold Nanoparticles Used as a Model Surface for Membrane Scaling in RO Wastewater Desalination.</a> Langmuir. 30, 15072-15082 (2014).</li><li>Zhang-Haagen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+Amyloid+Beta+Peptide+in+Hexafluoroisopropanol+Detected+by+Small+Angle+Neutron+Scattering.">Monomeric Amyloid Beta Peptide in Hexafluoroisopropanol Detected by Small Angle Neutron Scattering.</a> PLOS One. 11, e0150267 (2016).</li><li>Kaneko, F., Radulescu, A., Ute, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+small-angle+neutron+scattering+on+guest-exchange+processes+in+co-crystals+of+syndiotactic+polystyrene.">Time-resolved small-angle neutron scattering on guest-exchange processes in co-crystals of syndiotactic polystyrene.</a> J. Appl. Cryst. 47, 6-13 (2014).</li><li>Kohlbrecher, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+pressure+cell+for+small+angle+neutron+scattering+up+to+5000+MPa+in+combination+with+light+scattering+to+investigate+liquid+samples.">A high pressure cell for small angle neutron scattering up to 5000 MPa in combination with light scattering to investigate liquid samples.</a> Rev. Sci. Instr. 78, 125101 (2007).</li><li>Vavrin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+phase+diagram+of+an+adhesive+colloidal+dispersion+under+high+pressure:+A+small+angle+neutron+scattering,+diffuse+wave+spectroscopy,+and+light+scattering.">Structure and phase diagram of an adhesive colloidal dispersion under high pressure: A small angle neutron scattering, diffuse wave spectroscopy, and light scattering.</a> J. Chem. Phys. 130, 154903 (2009).</li><li>Kaneko, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Simultaneous+SANS/FTIR+Measuring+System.">Development of a Simultaneous SANS/FTIR Measuring System.</a> Chem. Lett. 44, 497-499 (2015).</li><li>Goerigk, G., Varga, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+upgrade+of+the+high-resolution+small+angle+neutron+scattering+instrument+KWS-3+at+FRM+II.">Comprehensive upgrade of the high-resolution small angle neutron scattering instrument KWS-3 at FRM II.</a> J. Appl. Cryst. 44, 337-342 (2011).</li><li>Desert, S., Thevenot, V., Oberdisse, J., Brulet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+very-small-angle+neutron+scattering+spectrometer+at+Laboratoire+L&#233;on+Brillouin.">The new very-small-angle neutron scattering spectrometer at Laboratoire L&#233;on Brillouin.</a> J. Appl. Cryst. 40, s471-s473 (2007).</li><li>Dewhurst, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D33+-+a+third+small-angle+neutron+scattering+instrument+at+the+Institute+Laue+Langevin.">D33 - a third small-angle neutron scattering instrument at the Institute Laue Langevin.</a> Meas. Sci. Technol. 19, 034007 (2008).</li><li>Zhao, J. K., Gao, K. Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extended+Q-range+small-angle+neutron+scattering+diffractometer+at+the+SNS.">The extended Q-range small-angle neutron scattering diffractometer at the SNS.</a> J. Appl. Cryst. 43, 1068-1077 (2010).</li><li>Takata, S., Suzuki, J., Shinohara, T., Oku, T., Tominaga, T., Ohishi, K., Iwase, H., Nakatani, T., Inamura, Y., Ito, T., Suzuya, K., Aizawa, K., Arai, M., Otomo, T., Sugiyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Design+and+q+Resolution+of+the+Small+and+Wide+Angle+Neutron+Scattering+Instrument+(TAIKAN)+in+J-PARC.">The Design and q Resolution of the Small and Wide Angle Neutron Scattering Instrument (TAIKAN) in J-PARC.</a> JPS Conf. Proc. 8, 036020 (2015).</li></ol>

The authors have nothing to disclose.

Soft matter and biophysical systems are typically characterized by structural correlations and interrelated microstructural and morphological levels that span a wide length scale, from nm to microns. To understand the mechanism of formation and evolution of the morphology of such systems and the relationship between their microscopic features and macroscopic properties, it is important to explore their microstructure over the entire length scale and under relevant environmental conditions (e.g., temperature, pressure, pH, humidity, etc.). Typically, small-angle scattering techniques with neutrons (SANS) or synchrotron X-rays (SAXS) are involved in such studies. The intensity drawback of neutrons versus synchrotron X-rays is compensated by the use of comparatively large &#916;&#955;/&#955;, which however leads to the worsening of the instrumental resolution. Nevertheless, SANS provides unique advantages due the possibilities offered by the contrast variation, especially between the hydrogen isotopes. Therefore, SANS is an experimental method specifically used in the study of soft matter and biophysical systems, for which it delivers unique structural and morphological information. Most of the SANS diffractometers worldwide21 work on the pinhole principle (Figure 2A), which enables the aimed low-Q resolution. Practically, all high-flux SANS diffractometers have a similar maximum flux on the order of 1 x 108 n cm-2 sec-1. Based on the relaxed wavelength resolution, the KWS-2 has a nearly doubled flux2, 4. Recently, very specialized SANS diffractometers became operational to serve with optimized characteristics for a specific range of applications, such as for investigations at very small scattering vectors22, 23. With the very recent commission of the specialized TOF-SANS diffractometers at steady-state reactors24 or spallation sources 25, 26, a massively-increased dynamic Q range in a given experimental setup and increased flexibility and optimization regarding the choice of the experimental resolution are offered. For the KWS-2 SANS diffractometer, a high level of versatility and performance required for very specific structural studies in the field of soft matter and biophysics is enabled on an otherwise classical SANS instrument. The optimization, flexibility, and spontaneity in designing and conducting complex studies, as supported by the described protocol, is achieved through the combination of the optimized experimental parameters (e.g., intensity, length scale, space resolution, and time resolution) and the complex sample environments. Using the multiple working modes enumerated in the Introduction and supported by the results presented in Figures 8-15, the KWS-2 enhances in an easy and practical manner the performance of a classical SANS diffractometer at a steady neutron source (reactor) beyond the conventional limits of such instruments.
This protocol presents the steps that a regular user must carry out to define and conduct a simple experimental program that involves only the investigation of samples at ambient thermodynamic conditions (temperature, pressure, relative humidity) and under static conditions (no kinetics of structure formation or transformation, no shear or flow). Several temperature-controlled holders or special sample environments (Table 1 and Supplementary Figure 19), such as pressure cells, rheometers, or humidity cells, are available and can be optimally installed and adjusted with special assistance from the instrument team. This protocol does not provide instructions about the settings and controls of such equipment. The definition and activation of external controllers require the use of another, more complex protocol. This protocol presents the case of working with quartz sample cells of a narrow rectangular shape (Figure 3). However, a broad range of cell geometries and types (Supplementary Figure 19) is offered to users, in order to provide increased flexibility and efficiency in conducting the experiments. In the case of using such cells, the present protocol may be followed with the adjustment of the parameters discussed in step 4.2.2. The measurement control software has been developed to offer the users increased flexibility in pursuing their scientific goals and optimization in the technical operation of the instrument. All adjustments and configurations of special functions and components of the instrument are done by the instrument team. The involvement of the scientific users in the configuration, definition, and utilization of the instrument is simplified and specifically limited only to those aspects that are in connection with the scientific issues of the experimental session. Configuration files are predefined in order to cover all special experimental issues, such as the positioning of special holders in the beam, the sample positioning in the beam (coordinates x, y, &#934;, and&#160;&#969; on the sample stage, rotation table, or cradle in Supplementary Figure 5), the adjustment of the detector and beam-stop positions for different wavelengths, the adjustment of the chopper parameters (frequency and opening window) for different wavelengths, the detection distances and aimed resolution, etc. Also, the current protocol does not describe how the real-time mode can be used on the KWS-2. The use of a more complex protocol is also required in order to conduct time-resolved SANS experiments.
Furthermore, this protocol presents how the measured data can be corrected for different scattering contributions from the instrument and the references and calibrated in order to obtain the sample&#39;s differential scattering cross section, d&#931;/d&#937;, expressed in cm-1. This quantity contains the entire structural and morphological information about the sample and is measured over a wide Q range corresponding to a broad length scale, over which structural correlations and interrelated size-levels characteristic of the investigated system appear. The scattering cross section d&#931;/d&#937; thus relates the intensity measured in a static scattering experiment at an angle &#920;, Is=f(&#920;) to the structural properties of the sample.
For the evaluation of d&#931;/d&#937; for a system of interest, besides the measurement of the system, additional measurements are needed in order to correct the data for any external scattering (i.e., environment, sample cell, solvent or buffer solution in the case of solute systems, etc.) and to calibrate the corrected data in absolute units8. The external background (sample cell or container), the reference sample (solvent or buffer solutions), the sample transmission (needed for the correct background subtraction and calibration of the corrected results in absolute units), the electronic background of the detector, the detector sensitivity (inhomogeneity in detector efficiency that is inherent for area detectors), and the normalized standard sample should also be measured. For the KWS-2, Plexiglas (PMMA) is used as standard sample. This is the so-called secondary standard and is periodically calibrated against a primary standard sample, which is vanadium. Vanadium delivers a very weak scattered intensity and requires very long measurement times for collecting the appropriate statistics; therefore, it is impractical for SANS purposes. The intensity collected from the sample of interest IS and from the standard sample ISt can be expressed as follows:
<img alt="Equation 1" src="/files/ftp_upload/54639/54639eq1.jpg" /> [1]
<img alt="Equation 1" src="/files/ftp_upload/54639/54639eq2.jpg" /> [2]
where I0 represents the incoming intensity (delivered by the collimation system), t is the thickness, A is the area exposed to the beam, T is the transmission, and &#916;&#968; is the solid angle at which a detection cell is seen from the sample position. If both sample and standard are measured in the same conditions with respect to the incoming beam (i.e., LC, AC and AS, and&#160;&#955; and &#916;&#955;/&#955;), I0 and A are the same and the solid angle is expressed as AD/LD (with AD representing the area of a detection cell). By dividing the two relations, the scattering cross-section of the sample is obtained as:
<img alt="Equation 1" src="/files/ftp_upload/54639/54639eq3.jpg" /> [3]
where the ISt is expressed as an average (the standard as an incoherent scattering system delivers a flat scattering pattern). The IS is obtained after correcting the measured intensity of the sample in the cell (container) with respect to the contribution of the empty cell IECell and the background on the detector for the closed beam, IB. The factor tStTSt(d&#931;/d&#937;)St, which contains the scattering and physical parameters of the standard sample, depends on the neutron wavelength&#160;&#955; and is typically known from the calibration of the standard sample. Thus, it is tabulated in the data reduction software4. The parameters and quantities in Eq. 3 that are known from calibration procedures and the definition of the experimental setup (tS, LD) form the so-called calibration factor k. The intensities and the sample transmission TS that appear in Eq. 3 must be measured. The data analysis program qtiKWS enables the correction, calibration, and radial averaging of the experimental data and the achievement of d&#931;/d&#937; for the investigated samples in a flexible and versatile working mode. The final results generated with the qtiKWS software are presented as tables with four columns: Q, I, &#916;I, &#916;&#963; where I represents d&#931;/d&#937; and&#160;&#916;&#963; is the Q resolution5.
From a practical point of view, with the KWS-2, combined SANS and USANS investigations can be carried out, with the advantage that the sample geometry and the thermodynamic conditions remain constant. Large morphologies that show multiple structural levels spanning a wide length-scale from nanometer to micrometer sizes can be investigated in a direct way, as shown in Figure 12. Besides the small-scale structural limit RC observed in the scattering curve measured in the conventional pinhole mode, by activating the lenses and the high-resolution detector, the large-scale structural limit LC of cylindrical core-shell micelles formed by the poly-(hexylene-oxide-co-ethylene-oxide) PHO10k-PEO10k diblock copolymer (fully protonated) in D2O14 could be observed at very low Q values in the extended Q-range working mode. The cylindrical micelles are characterized by a total thickness of about 300 &#197; and a length of about 7,000 &#197;, as revealed by the fit of the experimental results with the core-shell cylindrical form-factor9,14. Therefore, certain sensitive effects such as thermo-responsive gels or formation and growth of crystalline or partially-crystalline morphologies can be unambiguously explored with the KWS-2, unlike the classical approach of involving two or more different instruments and sample geometries.
As presented in Figure 11, correlated systems and ordered structures can be studied with adapted resolutions in a very flexible way, without spending time and effort on the installation of complex monochromatization systems, which would involve additional care and safety aspects. Moreover, by involving the chopper and the TOF data acquisition mode, monodisperse soft matter systems or complexes with low size polydispersity can be characterized very precisely at still-high intensities5.
The hindrances generated by the weak scattering due to use of highly-diluted systems or unfavorable contrast conditions can be overcome by the use of even higher intensities based on the larger beam size on the sample, while keeping the resolution. Figure 13A reports the scattering patterns from polystyrene particles of a radius of R = 150 &#197;, measured in the high-intensity mode using the lenses and a quadratic beam size ranging between 10 mm x 10 mm, the typical size used in the conventional pinhole mode, and 30 mm x 30 mm. Additionally, the result from a measurement with a round beam 50 mm in diameter (full lens size) is shown. In parallel, the normalized result obtained in the conventional pinhole mode is presented. Using 26 lenses with neutrons of &#955;= 7 &#197; and the same entrance aperture size AC as for the conventional pinhole mode (Figure 2B), a gain in intensity on the sample of about 12 times is obtained while keeping a constant beam size (resolution) on the detector, as shown in Figure 13B-C. The 27-lens system has a transmission of about 32% at ambient temperature. Cooled down to a temperature of 50 K, the lens transmission increases due the suppression of scattering on phonons in the lens material. The system of 26 parabolic lenses has a transmission of about 65% for a round beam size of 50 mm, when the beam goes through the entire volume of the lens, and of about 92% for a quadratic beam size of 10 mm x 10 mm, when only a very amount of lens material stays in the beam. The high-intensity mode with lenses offers benefits in the case of weak scattering that generally is encountered at a large detection distance and is particularly problematic in the case of weak contrast conditions. Additionally, when the sample is only stable over short period of times, the use of this mode represents a clear advantage, as is demonstrated elsewhere15.
On the other hand, in the case of biological systems, small sample volumes are typically available for experiments. Small biological molecules in physiological conditions with sizes of a few nanometers deliver weak scattering signals above the dominant scattering from the buffer solutions. Such signals can be measured with the KWS-2, benefiting from the high intensity of the instrument in the low-resolution setup of the pinhole mode, using short collimation lengths LC = 2 m or 4 m and short detection distances LD = 1 m, 2 m, or 4 m. Figure 14 presents the scattering patterns from beta amyloid protein (A&#946; 1-42, MW = 4.5 kDa) monomers in deuterated hexafluoroisopropanol dHFIP, as obtained after the correction for the scattering signal from the buffer was applied. A model fit of the data delivered a monomer size of about 16 &#177; 1 &#197;16. A long measurement time of several hours for each experimental condition (detection distance LD and sample type) was involved, although the measurements were carried out at short detection distances. The old detector, which has shown limitations regarding the count rate, hindered the use of short collimation distances LC, hence the use of the maximum flux at the instrument. With the commission of the new detection system enabling the use of the full neutron flux, such weak intensities will be measured in shorter times and with improved statistics in the future.
Finally, stimuli-sensitive effects can be studied in a flexible and easy manner by using the special ancillary equipment of the KWS-2. An example is reported in Figure 15, which shows the SANS patterns from Lysozyme protein in D2O buffer and from the buffer collected at different pressures. Special attention has been paid to the investigation of the background and forward scattering from the Lysozyme molecules, used in the performance test of the new pressure cell manufactured in-house by following a design done by PSI, Switzerland. The results were similar to those obtained by Kohlbrecher et al. in a similar study to test the original pressure cell model constructed there17, 18. With the KWS-2, further data has been acquired since a pressure of 5,000 bar has been reached. Evolution of the forward scattered intensity from the protein follows a linear behavior, as observed in the study at PSI, Switzerland18.

Soft and biological materials show a rich variety of morphologies that are characterized by features such as self-organization and self-assembly of elementary units to larger, complex aggregates. They also show cooperative interplay with a large number of degrees of freedom; weak interaction between the structural units, and thus high sensitivity to external fields; and spatiotemporal correlations that can span a broad range, from nanometers to millimeters and from nanoseconds to days. Because of the large range of relevant length- and timescales, the experimental characterization of the properties of these materials are very challenging. Scattering techniques with neutrons play an important role in the investigation of the structure, dynamics, and thermodynamic properties of such complex systems. As unique probes, neutrons offer the advantage of different interactions between the 1H and 2H (deuterium, D) hydrogen isotopes. The large difference in the coherent scattering length density between hydrogen and deuterium represents the basis of the contrast variation and contrast matching methods. As most of the soft matter and biological systems consist of hydrocarbons, hydrogen/deuterium (H/D) substitution offers the possibility to vary the coherent scattering length density of a compound over a broad range. With this technique, selected constituents in a complex system can be labeled by isotope exchange. Depending on its contrast-the squared difference between its scattering length density and that of the other components-selected components or regions within a complex soft matter or biophysical morphology can be made visible or invisible in the scattering experiment without chemically altering the system. Furthermore, neutrons are highly penetrating and can be used as non-destructive probes and for studying samples in special environments, where the contribution from the additional materials placed in the beam can be reliably measured and corrected for.
Elastic scattering experiments deliver information about the structure and morphology of a sample. The scattered intensity is measured in reciprocal space as a function of the momentum transfer Q, where Q=4&#960;/&#955; sin &#920;/2, with&#160;&#955; - the neutron wavelength and&#160;&#920; - the scattering angle; this is then translated into real space through an inverse Fourier transformation. Thus, large Q values relate to short length scales, with the inter-atomic correlations investigated by classical neutron diffraction (ND). At small Q values, large length scales can be explored by small-angle neutron scattering (SANS). Typically, single or assembling synthetic or natural macromolecules in solution, melt, film, or bulk samples are characterized over a wide length scale, from nanometer and micrometer sizes, via the application of the classical pin-hole SANS and the ultra-SANS (based on focusing or single-crystal diffractometry) techniques. However, the combination of different methods or facilities for achieving a complete structural characterization is sometimes difficult because of issues such as available amount of sample, stability of samples over long timescales, reproducibility of effects in special thermodynamic conditions, and the joint analysis of experimental data obtained in different experimental geometries. Moreover, the studies that deal with structures and fast structural changes that are characterized by high space or time resolution are very challenging, requiring very special experimental setups. Therefore, the development of highly versatile SANS instruments, where limits can be pushed beyond the typical configuration in an easy and practicable manner, is beneficial for meeting all special demands from the user community.
The SANS diffractometer KWS-2 (Figure 1), operated by the J&#252;lich Centre of Neutron Science (JCNS) at the Heinz Maier-Leibnitz Center (MLZ) in Garching, was originally a classical pinhole SANS instrument benefiting from a high neutron flux (Supplementary Figure 1) delivered by the FRM II neutron source1 and the dedicated guide system2-4. After repeated upgrades, the instrument was optimized for the exploration of a wide Q range, between 1x10-4 and 0.5 &#197;-1, providing high neutron intensities and an adjustable resolution. With the availability of specific sample environments and ancillary devices (Table 1), the instrument can be equipped to study soft matter and biophysical systems over a wide length scale, from nm up to &#181;m, through static measurements; it can also perform time-resolved investigations of structures and morphologies at equilibrium or under transformation due to kinetic processes, spanning a wide time scale between minutes and tens of milliseconds. In conventional working mode (Figure 2A), a Q range between 7x10-4 &#197;-1 and 0.5 &#197;-1 can be covered through the variation of the sample-to-detector distance and/or the wavelength. Therefore, structural levels and correlation effects on a length scale from 10 &#197; up to 9,000 &#197; can be inspected in real space (where the dimension is considered as 2&#960;/Q). The selection of the wavelength, between 4.5 &#197; and 20 &#197;, using a mechanical monochromator (velocity selector) that provides a wavelength spread &#916;&#955;/&#955;= 20%, the variation of the collimation conditions (collimation length LC and aperture openings, AC- the entrance aperture, following the last neutron guide segment in beam, and AS- the sample aperture, just in front of the sample) and of detection distance LD are done automatically, via computer control.
Considerable upgrades on the intensity on the sample, the instrument resolution, the minimum momentum transfer Qm, and the fast detection at high count rates in the MHz range were carried out recently, aiming to boost the instrument performance. During this process, the instrument was equipped with additional features.
There is a double-disc chopper5 with variable slit openings (Supplementary Figure 2) and time-of-flight (TOF) data acquisition mode. The chopper can be operated at a varying frequency fchopper between 10 Hz and 100 Hz and at angular openings of the two chopper windows between angles of 0&#176; &#8804; &#916;&#966; &#8804; 90&#176; by changing the positioning of the two discs with respect to each other. The improvement in wavelength resolution &#916;&#955;/&#955; is achieved by shortening the opening time of the neutron guide &#964;w by decreasing &#916;&#966; and/or increasing fchopper. The resulting pulses recorded on the detector are split in an appropriate number of time channels that match &#964;w in width and are characterized by the aimed &#916;&#955;/&#955;.
There are also focusing elements made of magnesium fluorite MgF2 parabolic lenses6 with a diameter of 50 mm (Figure 1). 26 MgF2 lenses are grouped into three packages (4+6+16 lenses) that can be moved independently in the beam to achieve focusing conditions with different wavelengths &#955;= 7-20 &#197;. In order to increase the transmission by reducing the scattering on phonons in the lens material, the lenses are kept at 70 K using a special cooling system.
There is a secondary high-resolution position-sensitive scintillation detector with a 1 mm position resolution and a 0.45 mm pixel-size. The detector is typically placed in the tower on the top of the vacuum tank at a fixed distance LD = 17 m and can be vertically moved in or out of the beam (Figure 1). The main detector is parked in the end position of the tank at 20 m, while the secondary detector is moved in the beam when high-resolution investigations (low Q) using lenses are being performed4,7. The secondary detector is placed in one focal point of the lens system, while a small entrance aperture at LC = 20 m would be, in this case, in the other focal point.
There is a new main detection system that consists of an array of 144 3He tubes (with a global efficiency per tube of 85% for &#955; = 5 &#197;) and that defines an active detection area equivalent to 0.9 m2 (Figure 1). Innovative rapid readout electronics mounted in a closed case on the backside of the 3He tube frame improves the read-out characteristics and reduces background noise. The new system that replaced the old scintillation detector (6Li scintillator and an array of 8x8 photomultipliers, Figure 1) is characterized by an effective dead-time constant of 25 nsec and an overall count rate as high as 5 MHz at 10% dead-time for flat profiles. These features are due to the fact that the system contains independent channels operated in parallel, which is an advantage over systems that experience dead-time after an event. The much higher count rate shortens the measurement times and therefore increases the number of experiments that can be performed in the same time period.
With all these innovations, the instrument became a highly versatile tool that can address a broad range of structural studies by offering multiple working modes (Table 2) that can be selected and used in a direct and user-friendly manner. In the high-intensity mode (Figure 2B), up to twelve times the intensity gain compared to the conventional pinhole mode for the same resolution can be achieved with lenses by increasing the sample size. In the tunable resolution mode with chopper and TOF data acquisition, improved characterization of the scattering features within different Q ranges are enabled by the possibility to vary the wavelength resolution &#916;&#955;/&#955; between 2% and 20%5. In the extended Q-range mode (Figure 2C), using lenses and the secondary high-resolution detector, a Qm as low as 1 x 10-4 &#197;-1 can be achieved, which, in combination with the pinhole mode, permits the exploration of sizes over a continuous length scale from the nm to the micron range. The use of a chopper for narrowing &#916;&#955;/&#955; provides accurate beam characteristics by avoiding gravity and chromatic effects when using the lenses. In the real-time mode, by exploiting the high intensity and external triggering of data acquisition by sample environments, structural changes can be resolved with time resolutions down to 50 msec. By improving the wavelength resolution down to &#916;&#955;/&#955;= 5% with the chopper, time resolutions as good as 2 msec can be realized.
Here, we present in detail a protocol on how typical experiments are conducted on the KWS-2 in its different working modes and how structural information from the investigated samples can be obtained from the collected data through data reduction. In this demonstration, we will use SANS to characterize several sizes of standard particle solutions and one highly-concentrated polymer micellar solution in order to show how size and order can be studied over broad ranges in a flexible and efficient way with the KWS-2 during one experimental session. Polystyrene spherical particles with different sizes (radii of R = 150, 350, 500, 1,000, and 4,000 &#197;) and a size polydispersity of &#963;R <img alt="Equation 5" src="/files/ftp_upload/54639/54639eq5.jpg" /> 8% are dispersed in a water solution (a mixture of 90% D2O and 10% H2O) at a volume fraction of 1%. Micelles formed by the C28H57-PEO5 diblock copolymers in D2O at a concentration of 12% show an ordered structure.

<table><tbody><tr><td>heavy water D&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>151882</td><td></td></tr><tr><td>heavy water D&lt;sub&gt;2&lt;/sub&gt;O/H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>151882</td><td>90% D&lt;sub&gt;2&lt;/sub&gt;O and 10% H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>3000 Series Nanosphere Size Standards (polystyrene)</td><td>Thermo Scientific</td><td>3030A</td><td>90% D&lt;sub&gt;2&lt;/sub&gt;O and 10% H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>3000 Series Nanospher Size Standards (polystyrene)</td><td>Thermo Scientific</td><td>3070A</td><td>90% D&lt;sub&gt;2&lt;/sub&gt;O and 10% H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>3000 Series Nanosphere Size Standards (polystyrene)</td><td>Thermo Scientific</td><td>3100A</td><td>90% D&lt;sub&gt;2&lt;/sub&gt;O and 10% H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>3000 Series Nanospher Size Standards (polystyrene)</td><td>Thermo Scientific</td><td>3200A</td><td>90% D&lt;sub&gt;2&lt;/sub&gt;O and 10% H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>3000 Series Nanosphere Size Standards (polystyrene)</td><td>Thermo Scientific</td><td>3800A</td><td>90% D&lt;sub&gt;2&lt;/sub&gt;O and 10% H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>diblock copolymer C&lt;sub&gt;28&lt;/sub&gt;H&lt;sub&gt;57&lt;/sub&gt;-PEO5k</td><td>synthesized in house</td><td></td><td>in D&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Quartz Cells 110-QX</td><td>Hellma analytics</td><td>110-1-46</td><td></td></tr><tr><td>Aluminum cuvette-holder</td><td>manufactured in house</td><td></td><td>for measurements at ambient temperature</td></tr><tr><td>screwdriver</td><td></td><td></td><td></td></tr><tr><td>Allen keys</td><td></td><td></td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2 at Jo

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

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. ...

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12.2K Views.  Forschungszentrum Jülich GmbH. Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to µm that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

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

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 ...

Engineering

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Chemistry

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Biochemistry

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Studying Large Amplitude Oscillatory Shear Response of Soft Materials

We investigate the sequence of physical processes exhibited during large amplitude oscillatory shearing (LAOS) of polyethylene oxide (PEO) in dimethyl sulfoxide (DMSO) and xanthan gum in water &#8212; two concentrated polymer solutions used as viscosifiers in foods, enhanced oil recovery, and soil remediation. Understanding the nonlinear rheological behavior of soft materials is important in the design and controlled manufacturing of many consumer products. It is shown how the response to LAOS of these polymer solutions can be interpreted in terms of a clear transition from linear viscoelasticity to viscoplastic deformation and back again during a period. The LAOS results are analyzed via the fully quantitative Sequence of Physical Processes (SPP) technique, using free MATLAB-based software. A detailed protocol of performing a LAOS measurement with a commercial rheometer, analyzing nonlinear stress responses with the freeware, and interpreting physical processes under LAOS is presented. It is further shown that, within the SPP framework, a LAOS response contains information regarding the linear viscoelasticity, the transient flow curves, and the critical strain responsible for the onset of nonlinearity.

We present a detailed protocol outlining how to perform nonlinear oscillatory shear rheology on soft materials, and how to run the SPP-LAOS analysis to understand the responses as a sequence of physical processes.

We investigate the sequence of physical processes exhibited during large amplitude oscillatory shearing (LAOS) of polyethylene oxide (PEO) in dimethyl ...

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 ...

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 ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

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

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