MBS acknowledges the support of the MI / CNRS under the contract Instrumentation at the limits 2014-2015. NJ acknowledges CEA&#39;s International Doctoral Research Program (Irtelis) for the PhD Fellowship. MBS and SJ acknowledge funding from the European Union&#39;s Horizon 2020 Research and Innovation Programme under Marie Sk&#322;odowska-Curie grant agreement number 722687. MBS, SJ, and NJ thank LIPhy (UGA) for the clean room establishment for microfabrication experiments. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

The authors have nothing to disclose.

A microfluidic device has been developed for on-chip protein crystallization with the microdialysis method and in situ X-ray diffraction experiments at room temperature. NOA 81 chips integrating RC dialysis membranes of any MWCO in order to use microdialysis for on-chip protein crystallization can be fabricated. Fabrication materials with a relatively high X-ray transparency were used, rendering the chips compatible for in situ protein crystallography. The fabrication materials that compose the compartment for protein crystallization of the device (PMMA, Kapton, RC dialysis membrane) were evaluated to generate low background noise. Specifically, the background noise generated by the dialysis chip is mainly observed at low resolution (&#62; 6 &#197;) and does not affect the treatment of high-resolution diffraction data of the large lysozyme crystals required for protein structure determination. The automation of the data collection is amplified using a 3D printed support that can be mounted directly in macromolecular crystallography beamlines and carry up to three microchips simultaneously. This way, manual harvesting and manipulation of the fragile protein crystals is avoided. Moreover, the data collection takes place at room temperature, avoiding the need for cryoprotection which can be related to conformational changes from the native protein structure2,3.
The use of microdialysis as a method to grow crystals on-chip allows to accurately monitor and control the crystallization process. As discussed in the introduction, most of the conventional protein crystallization methods have been implemented using microfluidic devices11,14. However, the advantages of dialysis for protein crystallization had not yet been fully exploited at the microscale. On-chip microdialysis provides the possibility to study phase diagrams and perform screening and optimization of crystallization conditions with the same protein sample19. For the prototypes presented in this work, the protein consumption per chip is limited down to 0.1 or 0.3 &#181;L. Based on the experimental work so far, the most critical steps of the protocol do not derive from the chips&#39; fabrication procedure but from the crystallization process. The fabrication protocol includes many steps but it is straightforward and enables the fabrication of numerous devices (20 to 30 chips) in a single day in the clean room, with relatively inexpensive materials. However, the on-chip crystallization of proteins can be a delicate procedure due to the intrinsic stochastic nature of nucleation and crystal growth, especially in the microscale. A case study has been described, where well-established conditions were used for the crystallization of lysozyme that yielded robust, well-defined crystals suitable for in situ X-ray diffraction data collection. Nevertheless, difficulties may arise by the use of more challenging protein targets, such as membrane proteins, where the crystallization medium is much more complicated, phase diagrams are not known and well-working crystallization conditions are not yet well established. The dialysis chip offers the possibility to surpass these difficulties and study phase diagrams on-chip, without disposing the valuable and frequently costly protein sample, by merely exchanging the crystallization solution within the microfluidic channel.
The versatility of the microfluidic devices stems from exploiting microdialysis for on-chip protein crystallization in order to reversibly control crystallization conditions and map concentration and temperature varied phase diagrams using low protein volume. Moreover, the device is compatible with in situ X-ray diffraction experiments and the prototyping of the devices is inexpensive and rapid. Numerous, isomorphous crystals of soluble and membrane proteins (in preparation) can be grown on-chip and it is expected that all these features can be utilized for serial X-ray crystallography studies of challenging protein targets at synchrotron and XFEL facilities. Finally, performing on-chip and in situ time-resolved studies is a future possibility that could be of significant interest to the crystallographic community. Therefore, by growing crystals on the dialysis chip and introducing the reagents into the microfluidic channel, either manually (using a syringe) or automatically (with a pressure-control fluid system or a syringe-pump), future efforts will focus on proving that the microfluidic chips can be successfully used to trigger time-resolved experiments on synchrotron beamlines.

Elucidating the three-dimensional (3D) structure of biological macromolecules is an unceasing pursuit in structural biology where X-ray crystallography remains the principal investigation technique. Applied for unraveling the structural details of complex macromolecules, such as proteins, it aims at facilitating the understanding of their mechanisms of actions and their involvement in various biological functions. Powerful X-ray sources at synchrotrons and X-ray free-electron lasers (XFELs) provide all the tools required for a deeper insight into the proteins&#39; structure at near atomic resolution. Despite the advantages that come along with the use of X-rays for structural studies, there are intrinsic limitations to X-ray radiation and the crystallization process itself. Radiation damage provoked by high X-ray flux and long exposure times of the protein crystal in front of the X-ray beam are restrictive parameters that crystallographers have to surpass using cryogenic cooling1. However, finding the optimal cryocooling conditions can be laborious since conformational changes from the native protein structure or artifacts can be concealed2,3. Moreover, recent studies indicate that performing diffraction experiments at room temperature leads to lower specific radiation damage4. Another bottleneck in structural biology is the acquisition of well-diffracting crystals with a sufficient size5. Small crystals are easier to produce, especially in the case of membrane proteins, but are more susceptible to radiation damage even under cryocooling conditions because a high radiation dose must be directed in a smaller volume compared to the case of larger protein crystals6. The novel approach of serial crystallography7,8 at synchrotrons and XFELs can circumvent the restrains of radiation damage and at the same time exploit smaller crystals (200 nm to 2 &#181;m)7 by merging data sets from multiple, isomorphous and randomly oriented protein crystals and profiting from the associated technological advances such as femtosecond pulses, shorter exposure times and micro-focused X-ray beams5,7,9,10.
Microfluidic technology is valuable to X-ray crystallography, exhibiting manifold advantages for the crystallization of biological macromolecules and their structural investigation. Conducting crystallization experiments in microfluidic devices requires small volumes of protein sample, therefore constraining the production cost of these high-valued bio macromolecules and facilitating high-throughput screening and optimization of numerous crystallization conditions. Moreover, the inherent large surface area-to-volume ratio at the microfluidic scale and diffusion-limited transport phenomena enable fine control over flows and temperature or concentration gradients11,12,13,14, rendering microfluidic devices suitable for growing uniformly sized crystals and exploring phase diagrams15,16,17,18,19. Moreover, microfluidic tools display a distinctive potential to address another hurdle in protein crystallography, which is the sample delivery, and the necessity to handle and harvest protein crystals prior to their use for X-ray diffraction experiments. The method of on-chip and in situ X-ray crystallography eliminates the crystal manipulation and the potential deterioration of crystal quality prior to data collection. A wide range of microfluidic chips compatible for in situ X-ray protein crystallography have been designed, developed, and tested by many research groups confronting the related restrictions arising from the nature of the microfabrication materials and their interactions with X-rays14,19,20,21,22,23. The fabrication materials must be optically transparent, biologically inert and demonstrate high transparency to X-ray radiation and an optimal signal-to-noise ratio during data collection.
Most of the crystallization methods applied in conventional protein crystallography24,25 have also been implemented at the microfluidic scale11,14 for on chip crystallization and in situ X-ray diffraction analysis. Simple, hybrid, or multi-layered microfluidic apparatus incorporating vapor diffusion26, evaporation27, free interface diffusion (FID)28, microbatch26, or even seeding29 have been used to crystallize soluble and membrane proteins. High throughput screening and optimization of crystallization conditions can be achieved30,31 in well-based32, droplet-based33, or valve-actuated34 devices. In situ X-ray diffraction experiments of challenging protein targets at room temperature have been conducted in microchips fabricated from various materials such as PDMS (polydimethylsiloxane), COC (cyclic olefin copolymer), PMMA (poly(methyl methacrylate))21,22,26,28,29, graphene films23, Kapton35, epoxy glue6, or NOA (Norland Optical Adhesive)19 and the materials&#39; transparency to X-ray radiation and their contribution to background noise have been evaluated. Moreover, microchips have been designed to couple the in situ and the serial data collection strategies in a single tool for X-ray protein crystallography experiments at synchrotron sources23,35,36 and XFELs7.
Room temperature&#160;in situ data collection has also been implemented in various delivery methods and devices. For example, Nogly et al.54 used a lipidic cubic phase (LCP) injector in order to study the structure of the light-driven photon pump bacteriorhodopsin (bR) by serial femtosecond crystallography (SFX) using an XFEL source. The crystal structure of bR was solved to 2.3 &#197; resolution, demonstrating the compatibility of an LCP injector with time-resolved serial femtosecond crystallography (TR-SFX). Baxter et al.55 designed a high-density multi-crystal grid, fabricated by a 100 or 200 &#181;m thick polycarbonate plastic with laser-cut holes of various sizes. An additional 5 &#181;m thick polycarbonate film can be fixed to one side of the grid when using the device for sitting- or hanging-drop crystallization experiments. This high-density grid can be used in multiple ways as crystals can be loaded directly onto the ports of the device or crystals can be grown on the device by vapor diffusion or the LCP method. Moreover, the grid can be adjusted in a standard magnetic base and used for in situ X-ray data collection at cryogenic or room temperature conditions. More recently, Feiler et al.56 developed a sample holder for macromolecular in situ&#160;X-ray crystallography at cryogenic and ambient temperature with minimal background noise contribution. Specifically, the holder comprises of a plastic support, a transparent COC foil and a microporous structured polyimide foil. It was designed to replace the commonly used cover slides for setting up crystallization drops, while allowing in-place manipulation such as ligand soaking, complex formation, and cryogenic protection without opening the crystallization drop or manually handling the crystals. Moreover, the sample holder can be removed from the crystallization plate and placed onto a magnetic base for in situ data collection at standard goniometer-based beamlines. For ambient temperature data collection, the COC foil is removed prior to the experiment and only the 21 &#181;m-thick polyimide foil contributes to background scattering, which in this case is minimal. These examples compose only a small fraction of the ongoing research and the multitude of versatile microchips developed for X-ray protein crystallography.
However, the dialysis protein crystallization method has not been widely incorporated within microfluidics. Dialysis is a diffusion-based method aiming for the equilibration of precipitant concentration through a semi-permeable membrane in order to approach the nominal concentration for protein crystallization and enables precise and reversible control over the crystallization conditions24. The Molecular Weight Cut-Off (MWCO) of the semi-permeable dialysis membrane can be chosen depending on the molecular weight of the macromolecule and the precipitants to allow the diffusion of small precipitant molecules while retaining the macromolecule of interest. Due to the reversibility of the dialysis process, it can be used in combination with temperature control to decouple and optimize nucleation and crystal growth independently37 for investigating phase diagrams by altering the precipitant concentration while using the same protein sample. The integration of membranes in microfluidics is reviewed by de Jong et al.38 and the case studies in biology implanting dialysis into microchips can be principally listed in sample preparation, concentration or filtration applications39,40,41,42 or cell-related studies43,44. Pervaporation through PDMS was used by Shim et al.37 to study the nucleation and growth of xylanase in various conditions. Water permeated through the 15 &#181;m thick PDMS membrane into the protein reservoir of the microfluidic device, subsequently altering the protein and precipitant concentration.
The protocol developed by Junius et al.19,45 for the fabrication of a microfluidic chip compatible for both on-chip protein crystallization via microdialysis and in situ X-ray diffraction experiments at room temperature is presented. The protocol for the device fabrication is directly inspired by the pioneering work accomplished by Studer and coworkers12,46 for micro-patterned stickers of photo-curable thiolene-based resin NOA 81 embedding commercially available membranes, using soft imprint lithography. An innovative modification of the method resulted in microchips enabling the use of microdialysis to accurately monitor and control the experimental parameters for the on-chip growth of protein crystals and simultaneously exploit&#160;the advantages of microfluidics, such as reduced consumption of protein samples per experiment (&#60;1 &#181;L). In a previous work, the principles of dialysis applied to a macro-scale system (typical volume &#62;20 &#181;L) for screening and optimizing crystallization conditions by mapping temperature-precipitant concentration phase diagrams were demonstrated47. In this work, a protocol is described for producing dialysis microchips incorporating regenerated cellulose (RC) dialysis membranes of different MWCO in order to perform crystallization assays on-chip and in situ X-ray diffraction data collection. The materials comprising the microchips have been evaluated for their transparency to X-rays19 and the devices can be set directly in front of the X-ray beam for room temperature in situ diffraction experiments, excluding the manual handling and minimizing the degradation of fragile protein crystals. In a case study, hen egg-white lysozyme crystals were grown on-chip via microdialysis generating a uniformly sized population. The microchip was then mounted in front of the X-ray beam with a 3D-printed support19 and complete in situ diffraction data sets were collected at room temperature from multiple, isomorphous crystals, demonstrating the high potential and relevance of the chips for synchrotron serial crystallography studies of challenging macromolecular targets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 in wafer</td>
			<td>Silicon Materials Inc.</td>
			<td></td>
			<td>Silicon wafer</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Minispin</td>
			<td>Bench-top centrifuge</td>
		</tr>
		<tr>
			<td>CleWin 3.0</td>
			<td>WieWeb software</td>
			<td></td>
			<td>Designing software</td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Devcon</td>
			<td></td>
			<td>5 minutes epoxy glue</td>
		</tr>
		<tr>
			<td>Fluidic connectors</td>
			<td>Cluzeau Info Lab</td>
			<td>N-333</td>
			<td>NanoPort kit for 1/16&#34; OD tubing</td>
		</tr>
		<tr>
			<td>Hen egg-white lysozyme</td>
			<td>Roche</td>
			<td>10 837 059 001</td>
			<td>Lyophilized protein powder</td>
		</tr>
		<tr>
			<td>High-vacuum silicone grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554</td>
			<td>Dow Corning&#160;high-vacuum silicone grease</td>
		</tr>
		<tr>
			<td>HMDS</td>
			<td>Sigma-Aldrich</td>
			<td>440191</td>
			<td>Silane, chemical</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Sawatec</td>
			<td>HP-200-Z-HMDS BM</td>
			<td>Hot plate</td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Kapton tape</td>
			<td>DuPont</td>
			<td></td>
			<td>Polyimide tape</td>
		</tr>
		<tr>
			<td>Mask aligner</td>
			<td>SUSS MicroTec</td>
			<td>MJB4</td>
			<td>Mask aligner, UV source</td>
		</tr>
		<tr>
			<td>Membrane filter</td>
			<td>Millipore</td>
			<td>GSWP04700</td>
			<td>0.22 &#956;m pore size filter</td>
		</tr>
		<tr>
			<td>Microscope glass slide</td>
			<td>Fisher Scientific</td>
			<td>12164682</td>
			<td>3 x 1 in glass slides</td>
		</tr>
		<tr>
			<td>NOA81</td>
			<td>Norland Products Inc.&#160;</td>
			<td>NOA81</td>
			<td>Photocurable resin</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Memmert</td>
			<td></td>
			<td>Oven</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>P6543</td>
			<td>Parafilm M roll size 20&#160;in. &#215; 50&#160;ft</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td>Silicone</td>
		</tr>
		<tr>
			<td>PEG 400</td>
			<td>Hampton Research</td>
			<td>HR2-603</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sigma-Aldrich</td>
			<td>P5731</td>
			<td>100 x 15 mm</td>
		</tr>
		<tr>
			<td>PGMEA</td>
			<td>Sigma-Aldrich</td>
			<td>484431</td>
			<td>Developer</td>
		</tr>
		<tr>
			<td>Plasma equipment</td>
			<td>Diener Electronic</td>
			<td>ZEPTO</td>
			<td>Plasma treatment</td>
		</tr>
		<tr>
			<td>PMMA</td>
			<td>Goodfellow</td>
			<td>137-745-63</td>
			<td>PMMA sheets 150x150 mm, 0.175 mm thickness</td>
		</tr>
		<tr>
			<td>Pressure driven system</td>
			<td>Elveflow</td>
			<td>OB1 MK3+</td>
			<td>Pressure/vacuum controller</td>
		</tr>
		<tr>
			<td>PTFE tubing</td>
			<td>Elveflow/Darwin microfluidics</td>
			<td>LVF-KTU-15</td>
			<td>PTFE tubing roll 1/16&#34; OD X 1/32&#34; ID</td>
		</tr>
		<tr>
			<td>RC dialysis membrane</td>
			<td>Spectra/Por</td>
			<td></td>
			<td>Various MWCOs</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-Morton</td>
			<td></td>
			<td>Carbon steel surgical blades</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Solidworks</td>
			<td>Dassault Systemes</td>
			<td></td>
			<td>3D-CAD designing software</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>SPS</td>
			<td>Spin150</td>
			<td>Wafer spinner</td>
		</tr>
		<tr>
			<td>SU-8 3000 series</td>
			<td>MicroChem Corp.</td>
			<td>SU-8 3050</td>
			<td>Photoresist</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 mL Luer-Lok syringe</td>
		</tr>
		<tr>
			<td>UV crosslinker</td>
			<td>Uvitec</td>
			<td>CL-508</td>
			<td>UV crosslinker</td>
		</tr>
	</tbody>
</table>

crystallization of proteins on chip by microdialysis for in situ x-ray diffraction studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Watch this Scientific Journal Video about Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies at JoVE.com

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

crystallization-proteins-on-chip-microdialysis-for-situ-x-ray

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Chemistry

6.4K Views.  Université Grenoble Alpes, CEA, CNRS, IBS. This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

Video: Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

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 ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

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 ...

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 ...

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

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 ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...