We acknowledge Gebhard Schertler, Rafael Abela and Chris Milne for supporting the use of high viscosity injectors at the PSI. Richard Neutze and his team are acknowledged for discussions on time-resolved crystallography and sample delivery using high viscosity injectors. For financial support, we acknowledge the Swiss National Science Foundation for grants 31003A_141235, 31003A_159558 (to J.S.) and PZ00P3_174169 (to P.N.). This project has received funding from the European Union&#39;s Horizon 2020 research and innovation program under the Marie-Sklodowska-Curie grant agreement No 701646.

1. Protein Crystal Sample Preparation
<ol>
	<li>About 30 min before the sample is to be injected, load 50 &#181;L of crystal-laden monoolein based LCP in a 100 &#181;L syringe.</li>
	<li>For injection at atmospheric pressure: Load 10 &#181;L of liquid paraffin into the back of a second syringe. Holding the syringe vertically, expel the air bubbles from the syringe.
	<ol>
		<li>For injection into vacuum environment: Load 5 &#181;L of MAG 7.9 and 5 &#181;L of liquid paraffin into the back of a second syringe. Holding the syringe vertically, expel the air bubbles from the syringe.</li>
	</ol>
	</li>
	<li>Connect the syringe with paraffin/MAG 7.9 to a standard syringe coupler, purge the air from the coupler by gently pressing on the plunger until a small volume (&#60;1 &#181;L) of paraffin/lipid is visible at the tip of the coupler needle.</li>
	<li>Connect the sample syringe to the syringe coupler, taking care to not introduce any air into the sample. Mix in the lipid/paraffin by passing the sample material through the coupler multiple times.</li>
	<li>Load 20 &#181;L of premixed LCP (27% PEG, 100 mM Sorensen pH 5.6 + MO) into another 100 &#181;L syringe. Remove air bubbles as necessary.</li>
	<li>Remove the empty syringe from the coupler and attach the premixed LCP to the crystal containing syringe using a standard syringe coupler. Pass the sample through the coupler 100 times. 
	NOTE: Sample mixing may heat the sample slightly46. Mixing should be done at a slow rate where the temperature of the sample can be held reasonably constant.</li>
	<li>Inspect the sample for transparency against a source of light. If a clear homogeneous LCP has formed, go to step 1.9.</li>
	<li>To bring the sample into the cubic phase, add 3 &#181;L of monoolein and mix 50 times (as described above). Repeat this procedure just until a transparent phase has formed to avoid an excess of monoolein. 
	NOTE: The formation of the LCP is temperature dependent47 and best results are achieved slightly above 20 &#176;C. The amount of additional monoolein needed will depend on the volume of residual precipitant solution carried over from crystallization.</li>
	<li>As a preliminary test for sample stiffness (as expected from the LCP phase) and extrude-ability, detach the empty syringe from the syringe coupler and, holding the syringe vertically, squeeze a small amount of sample (&#60;2 &#181;L) through the coupler. If the extruded sample forms an upright cylinder, then the sample is ready for extrusion testing.</li>
	<li>Adjust the total volume of the sample to 100 &#181;L by adding more premixed LCP (as in step 1.5).</li>
	<li>Attach the sample syringe and two empty syringes to the three-way syringe coupler (purging the air from the coupler as before). Mix at least 50 times (or until homogeneous) by passing half of the sample into the second syringe and then pressing both halves of the sample into the third syringe simultaneously.</li>
	<li>Place the syringe containing the mixed sample under a stereo microscope to verify a homogenous distribution of crystals.</li>
</ol>
2. Testing Sample Extrusion Using a High-Speed Camera Setup
<ol>
	<li>Determining experimental parameters.
	<ol>
		<li>Select the nozzle size for the test. 
		NOTE: Nozzle sizes are typically 50 or 75 &#181;m internal diameter (ID), but any size from roughly 30-100 &#181;m ID may be considered. Selection is based on a balance between mean clogging time, background scattering, sample consumption, and hit-rate.</li>
		<li>Calculate optimum flow speed based on experimental laser spot size (1/e2 diameter), and the data collection scheme (e.g., interleaved light and dark) that will be used at the XFEL.</li>
		<li>Calculate the desired flow rate (<img alt="Equation 1" src="/files/ftp_upload/59087/59087eq1.jpg" />) of the sample from the optimum flow speed (<img alt="Equation 2" src="/files/ftp_upload/59087/59087eq2.jpg" />) and the selected nozzle diameter (<img alt="Equation 3" src="/files/ftp_upload/59087/59087eq3.jpg" />): 
		<img alt="Equation 4" src="/files/ftp_upload/59087/59087eq4.jpg" /> 
		Determine the flow rate (<img alt="Equation 5" src="/files/ftp_upload/59087/59087eq5.jpg" />) that should be entered at the HPLC pump based on the amplification factor (<img alt="Equation 6" src="/files/ftp_upload/59087/59087eq6.jpg" />) of the injector. 
		<img alt="Equation 7" src="/files/ftp_upload/59087/59087eq7.jpg" /> 
		Calculate the maximum pressure (<img alt="Equation 8" src="/files/ftp_upload/59087/59087eq8.jpg" />) from the rated pressure of the nozzle fittings (<img alt="Equation 9" src="/files/ftp_upload/59087/59087eq9.jpg" />) and the amplification factor of the injector (<img alt="Equation 10" src="/files/ftp_upload/59087/59087eq10.jpg" />): 
		<img alt="Equation 11" src="/files/ftp_upload/59087/59087eq11.jpg" /></li>
	</ol>
	</li>
	<li>Setup of the high viscosity extrusion injector for offline use as shown in Figure 1. 
	NOTE: The injector functions by means of hydraulic extrusion powered by an HPLC pump, and uses a co-flowing helium gas sheath controlled by a gas regulator. The pump and regulator setup are not discussed in this protocol. See chapter four in James (2015)48 for a detailed manual.
	<ol>
		<li>Purge the pump and all water lines to ensure the flowrates are accurate. Purge the hydraulic stage of the injector.</li>
		<li>Mount the injector (or the camera) on a three-axis stage to facilitate framing and focus for the high-speed videos. Leave space around the injection point for the objective lens, illumination, reflective screen, and a small beaker to catch the spent sample.</li>
		<li>Construct a &#8220;dummy nozzle&#8221; (an empty reservoir with a nozzle attached) and install it on the injector to facilitate positioning, focus, and illumination.</li>
		<li>Mount the high-speed camera with the nozzle tip near the focal point of the objective.</li>
		<li>Position the reflective screen behind the injector and adjust the illumination to front-light the nozzle.</li>
		<li>Turn on and connect to the camera with the supplied software. With a live video running for visual feedback, position the nozzle tip in the center of the frame, and bring it into focus with the three-axis stage.</li>
		<li>Adjust the illumination until the nozzle is clearly visible and the background is evenly illuminated.</li>
	</ol>
	</li>
	<li>Configuring the camera to record high-speed time-lapse video.
	<ol>
		<li>Set framerate to 1000 fps. Set resolution to 512 x 512 pixels.</li>
		<li>With the exposure time now set by the frame rate, adjust the illumination level until the nozzle is visible (i.e., not under- or overexposed). Reposition the nozzle tip so that it is centered left to right and is located in the top third of the frame.</li>
		<li>Run any background correction or white balance operations.</li>
		<li>Set up the camera in time-lapse mode. Set interval to 30 s and repeats to 40 times, set the trigger mode to random (or random reset), and enter the number of frames to record to 1000.</li>
	</ol>
	</li>
	<li>Load the reservoir with 20 &#181;L of the test sample, and attach the capillary nozzle.</li>
	<li>Attach the filled reservoir to the injector. Attach the gas line to the port on the nozzle and start the gas flow.</li>
	<li>Manually advance the piston by opening the in-line valve and pressing the syringe. Close the valve when the piston makes solid contact with the sample in the reservoir.</li>
	<li>Program the pump with the calculated flow rate and set the maximum pressure to the calculated value (see step 2.1.3).</li>
	<li>Simultaneously start the pump and the camera recording.</li>
	<li>As the extrusion begins, adjust the gas pressure to increase stability. Increase gas pressure if the fluid forms a drop at the tip of the nozzle instead of a column. Decrease pressure if the extrusion is broken due to excessive shear from the gas flow, or is oscillating rapidly (whipping).</li>
	<li>Monitor the extrusion (10 min is usually enough to recognize a homogenous flow condition) and, when the pump pressure sharply ramps up near the expected end time, stop recording, shut off the pump, and vent the system pressure by opening the relief valve.</li>
	<li>Analysis of the video files 
	NOTE: Sample extrusion that is plainly inadequate does not require a detailed analysis. However, it is still useful to identify the failure mode so that the sample may be optimized.
	<ol>
		<li>Open the video file with the analysis software (e.g., Fiji49) and calibrate the measurement tools.</li>
		<li>Calibrate the measurements by selecting a line of known length in the video image with the line selection tool. Open the Set Scale window via Analyze | Set Scale and enter the known distance and the units of measurement. 
		NOTE: The known diameter of the nozzle provides an adequate calibration length for these measurements.</li>
		<li>Find a frame in the video where there is a trackable feature visible (e.g., a crystal) in the extrusion. Record the frame number.</li>
		<li>Advance the video to a frame where the same feature is visible but has moved from its position in the previous step. Record the frame number.</li>
		<li>Using the straight line measurement tool, measure the distance from the feature start and endpoint via Analyze | Measure. 
		NOTE: The longer the trackable distance, the fewer errors in measurement will affect the calculations</li>
		<li>Calculate the jet speed from the measured distance and the elapsed time (the number of elapsed frames divided by framerate.)</li>
		<li>Repeat steps 2.11.3-2.11.6 a few times for every segment of the video.</li>
		<li>Plot the data series as in Figure 2.</li>
	</ol>
	</li>
</ol>

The authors declare no conflicting interests.

The TR-SFX method with the viscous extrusion injector has proven to be a viable technique for structural dynamics studies of bacteriorhodopsin9,10 and photosystem II13 and now seems ready to study proteins driving other photo biological processes such as light-driven ion transport or sensory perception5,50. The protocols described above were designed to maximize the success of TR-S...

Serial femtosecond crystallography (SFX) is a structural biology technique that exploits the unique properties of X-ray free electron lasers (XFEL) to determine room temperature structures from thousands of micrometer-sized crystals while outrunning most of the radiation damage by the &#34;diffraction before destruction&#34; principle1,2,3.
In a time-resolved extension of SFX (TR-SFX), the femtosecond pulses from the XFEL are used to study structural changes in proteins4,5. The protein of interest is activated with an optical laser (or another activity trigger) just prior to being shot by the XFEL in a pump-probe setup. By precisely controlling the delay between pump and probe pulses, the target protein can be captured in different states. Molecular movies of structural changes over eleven orders of magnitude in time demonstrate the power of the new XFEL sources to study the dynamics of several protein targets6,7,8,9,10,11,12,13. Principally, the method joins the dynamic spectroscopic and static structural techniques into one, providing a glimpse into protein dynamics at near atomic resolution.
Simple systems for TR-SFX may contain an endogenous trigger of activation with a photo-sensitive component like retinal in bacteriorhodopsin (bR)9,10, the chromophores in photosystem II12,13, photoactive yellow protein (PYP)6,7 reversibly photoswitchable fluorescent protein11, or a photolyzable carbon monoxide in myoglobin8. Exciting variations of the technique still in development rely on mix and inject schemes14,15 to study enzymatic reactions or an electric field used to induce structural changes16. Given that XFEL sources have only been available for a few years and extrapolating past successes into the future, the method shows potential as a real game-changer with respect to our understanding of how proteins function.
Because biological samples are destroyed by a single exposure to a high power XFEL pulse, new approaches to protein crystallography were necessary. Among these procedures, the ability to grow large amounts of uniform microcrystals needed to be developed17,18,19. To enable data collection at an XFEL, these crystals must be delivered, discarded, and then renewed for each XFEL pulse. Given that XFELs fire usable pulses at 10-120 Hz, sample delivery must be fast, stable, and reliable, while also keeping the crystals intact and limiting consumption. Among the most successful solutions is a high viscosity micro-extrusion injector, which delivers a continiously streaming column of room temperature crystal-laden lipidic cubic phase (LCP) across the pulsed X-ray beam20. Randomly oriented crystals, embedded in the LCP stream, that are intercepted by the XFEL pulses scatter X-rays onto a detector where a diffraction pattern is recorded. LCP was a natural choice for a sample delivery medium as it is frequently used as a growth medium for membrane protein crystals17,21,22,23, yet other high viscosity carrier media24,25,26,27,28,29,30 and soluble proteins31 have also been used in the injector. SFX with the high viscosity injector has been successful during the structure determination of membrane proteins13,32 including G protein-coupled receptors (GPCRs)33,34,35,36,37, with data quality sufficient for native phasing38,39 while being both time and sample efficient. Currently, these injectors are being used more routinely for room temperature measurements at synchrotron sources28,30,40,41 as well as during the more technically demanding TR-SFX experiments at XFELs9,10,13,42.
Comparable TR-SFX experiments have been carried out using other injector types like liquid phase delivery in a flow focused nozzle6,7,12, however, this method requires protein amounts not available for many biologically interesting targets. For the determination of static structures using viscous extrusion an average consumption of 0.072 mg of protein per 10,000 indexed diffraction patterns in comparison to 9.35 mg for the liquid jet nozzles have been reported (i.e., about 130 times more sample efficient)20. The high viscosity injector has been shown to be a viable sample delivery device for TR-SFX while only sacrificing some of this sample efficiency43. In Nogly et al. (2018)10, for example, sample consumption was about 1.5 mg per 10,000 indexed patterns, which compares favorably to similar TR-SFX experiments using the PYP where average sample consumption was much higher with 74 mg of protein per 10,000 indexed patterns6. High viscosity injectors thus have clear advantages when the amount of protein available is limiting or when crystals are grown directly in LCP.
For TR-SFX using high viscosity injectors to yield the most reliable data several technical issues need to be addressed: the flow speed needs to remain above a minimum critical value; the hit-rate should be maintained at a level that does not render data collection slow (e.g., greater than 5%); and sample has to be delivered without excessive disruptions. Ideally, these conditions are already met long before a scheduled TR-SFX experiment to use available XFEL time as efficiently as possible. Pricipally, a slowdown in the LCP stream may allow probing crystals that were activated with more than one optical laser pulse and result in mixed active states, or probing pumped material when umpumped material is expected in the beam. An additional benefit of injection pre-testing is that downtime during data collection at an XFEL is minimized as time relegated to replacing clogged nozzles, changing out non-extruding samples, and other maintenance tasks is reduced.
Here, we present a method to optimize sample delivery for TR-SFX data collection with a high viscosity micro-extrusion injector. For simplicity, the described methods do not rely on access to an X-ray source, although work at a synchrotron beamline29 would provide further information on expected hit rates and crystal diffraction. Our protocols were developed to optimize experiments to capture retinal isomerization in the proton-pump bacteriorhodopsin10 and are carried out in two phases starting with preparing crystal samples for extrusion followed by monitoring the extrusion using a high-speed camera setup. In phase one, the crystal-laden LCP is mixed with additional LCP, low transition temperature lipids, or other additives to ensure the final mixture is suitable for delivery into the sample environment without clogging or slowing. A new three-way syringe coupler was developed to improve mixing performance and sample homogeneity. The second phase consists of an extrusion test recorded by a high-speed camera to directly measure the extrusion speed stability. Following the analysis of the video data, adjustments can be made to the sample preparation protocol to improve experimental outcomes. These procedures can be adapted to prepare other proteins for TR-SFX data collection, with minimal modifications, and will contribute to the efficient use of limited XFEL beamtime. With new XFEL facilities just starting their operation44,45 and the transfer of injector-based serial data collection methods to synchrotrons28,30,40,41, the next few years will surely continue to provide exciting new insights into the structural dynamics of an ever-wider range of protein targets.

<table border="0" cellpadding="0" cellspacing="0" style="width:745px;" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Mosquito LCP Syringe Coupling</td>
			<td style="width:175px;">TTP labtech store</td>
			<td style="width:101px;">3072-01050</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Hamilton Syringe 1710 RNR, 100 &#181;l</td>
			<td style="width:175px;">Hamilton</td>
			<td style="width:101px;">HA-81065</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Hamilton Syringe 1750 RNR, 500 &#181;l</td>
			<td style="width:175px;">Hamilton</td>
			<td style="width:101px;">HA-81265</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Monoolein</td>
			<td style="width:175px;">Nu-Chek Prep, Inc.</td>
			<td style="width:101px;">M-239</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">7.9 MAG</td>
			<td style="width:175px;">Avanti Polar Lipids Inc.</td>
			<td style="width:101px;">850534O</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">50% w/v PEG 2000</td>
			<td style="width:175px;">Molecular Dimensions</td>
			<td style="width:101px;">MD2-250-7</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Paraffin (liquid)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:101px;">1.07162</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">High speed camera</td>
			<td style="width:175px;">Photron</td>
			<td style="width:101px;"></td>
			<td style="width:224px;">Photron Mini AX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">High magnification lens</td>
			<td style="width:175px;">Navitar</td>
			<td style="width:101px;"></td>
			<td>12X Zoom Lens System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Three axis stage</td>
			<td style="width:175px;">ThorLabs</td>
			<td style="width:101px;">PT3/M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Fiber light</td>
			<td style="width:175px;">Thorlabs</td>
			<td style="width:101px;">OSL2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Fused silica fiber</td>
			<td style="width:175px;">Molex/Polymicro</td>
			<td style="width:101px;">TSP-505375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Lite touch ferrule</td>
			<td style="width:175px;">IDEX</td>
			<td style="width:101px;">LT-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">ASU high viscosity injector</td>
			<td style="width:175px;">Arizona State University</td>
			<td style="width:101px;"></td>
			<td>Purchasable from Uwe Weierstall (weier@asu.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">HPLC pump</td>
			<td style="width:175px;">Shimadzu</td>
			<td style="width:101px;">LC-20AD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Electronic gas regulator</td>
			<td style="width:175px;">Proportion Air</td>
			<td style="width:101px;">GP1</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving high viscosity extrusion of microcrystals for time-resolved serial femtosecond crystallography at x-ray lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The ideal starting material for the procedures described here (Figure 3) are high densities of microcrystals incorporated into viscous carrier medium for the injector. The procedure calls for about 50 &#181;L of crystal laden carrier for each preparation. These can be grown directly in LCP as with the bR9,10 used here, as an example (Figure 4), or prepared using crystals ...

Watch this Scientific Journal Video about Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers at JoVE.com

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...

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8.9K Views.  Paul Scherrer Institut. The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

Video: Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Sediment Core Extrusion Method at Millimeter Resolution Using a Calibrated, Threaded-rod

Aquatic sediment core subsampling is commonly performed at cm or half-cm resolution. Depending on the sedimentation rate and depositional environment, this resolution provides records at the annual to decadal scale, at best. An extrusion method, using a calibrated, threaded-rod is presented here, which allows for millimeter-scale subsampling of aquatic sediment cores of varying diameters. Millimeter scale subsampling allows for sub-annual to monthly analysis of the sedimentary record, an order of magnitude higher than typical sampling schemes. The extruder consists of a 2 m aluminum frame and base, two core tube clamps, a threaded-rod, and a 1 m piston. The sediment core is placed above the piston and clamped to the frame. An acrylic sampling collar is affixed to the upper 5 cm of the core tube and provides a platform from which to extract sub-samples. The piston is rotated around the threaded-rod at calibrated intervals and gently pushes the sediment out the top of the core tube. The sediment is then isolated into the sampling collar and placed into an appropriate sampling vessel (e.g., jar or bag). This method also preserves the unconsolidated samples (i.e., high pore water content) at the surface, providing a consistent sampling volume. This mm scale extrusion method was applied to cores collected in the northern Gulf of Mexico following the Deepwater Horizon submarine oil release. Evidence suggests that it is necessary to sample at the mm scale to fully characterize events that occur on the monthly time-scale for continental slope sediments.

An extrusion method using a calibrated threaded-rod is presented, which allows for mm scale subsampling of aquatic sediment cores. Millimeter-scale sampling is necessary to fully characterize recent event stratigraphy in sediment records.

Aquatic sediment core subsampling is commonly performed at cm or half-cm resolution. Depending on the sedimentation rate and depositional environment, ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

An Experimental Protocol for Femtosecond NIR/UV - XUV Pump-Probe Experiments with Free-Electron Lasers

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a free-electron laser. This includes methods to establish the spatial and temporal overlap between the optical and free-electron laser pulses during the experiment, as well as important aspects of the data analysis, such as corrections for arrival time jitter, which are necessary to obtain high-quality pump-probe data sets with the best possible temporal resolution. These methods are demonstrated for an exemplary experiment performed at the FLASH (Free-electron LASer Hamburg) free-electron laser in order to study ultrafast photochemistry in gas-phase molecules by means of velocity map ion imaging. However, most of the strategies are also applicable to similar pump-probe experiments using other targets or other experimental techniques.

This protocol describes the key steps for performing and analyzing pump-probe experiments combining a femtosecond optical laser with a free-electron laser in order to study ultrafast photochemical reactions in gas-phase molecules.

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...