Name: microfluidic chips for in situ crystal x-ray diffraction and in situ dynamic light scattering for serial crystallography
Uploaded: 2018-04-24T13:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography at JoVE.com

This work was supported by the PIER seed fund PIF-2015-46, the BMBF grants 05K16GUA and 05K12GU3, and the &#39;The Hamburg Centre for Ultrafast Imaging &#8211; Structure, Dynamics and Control of Matter at the Atomic Scale&#39; excellence cluster of the Deutsche Forschungsgemeinschaft (DFG). The work of authors affiliated with the Center for Free-Electron Laser Science was funded by the Helmholtz Association through programme oriented funds. The synchrotron MX data was collected at beamline P14 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany).

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The authors have nothing to disclose.

We fabricate microfluidic devices for in situ X-ray diffraction by patterning epoxy resin as filling material and polyimide foil as window material. Our procedure optimized various steps of the fabrication process over previous X-ray chip designs16,21. We reduced the window thickness and thereby the background scattering while also easing fabrication as fewer process steps are required. in situ crystallization using the described protocol has substantial benefits. It allows diffraction data collection at room-temperature and thereby excludes the need of cryo protection, which in some cases contains the risk of introducing artefacts in the protein structure. Furthermore, the crystals are not subject to physical stress, because the transfer of the crystals from their native environment can be avoided. Through this procedure, the crystals maintain their highest quality and do not suffer from any treatment.&#160;
In our experience, the most critical steps within the protocol revolve around controlling the crystallization process. The parameters to obtain X-ray suitable crystals with appropriate dimensions need to be identified empirically and cannot be taken directly from vapor diffusion experiments. Using identical concentrations of protein and precipitant did not always result in crystals in different chips, or at times in different wells within the same chip. This indicates that all factors influencing crystal nucleation and growth should be considered carefully, such as mother liquor composition or crystallization kinetics (through the evaporation trajectory). As larger crystals diffract to higher resolution, suitably large crystals are ideally grown. The process of crystal nucleation and growth may be followed with DLS measurements. Adjusting the laser focus inside the ~50 &#181;m thin crystallization compartments of the chip can be challenging and may require careful manual alignment. By using wells deeper than 100 &#181;m, laser auto-alignment was feasible and reliable, such that multiple wells could be monitored through automated acquisition schemes.
The polyimide based X-ray chips produce only a low background and we demonstrate the suitability of these devices for routine X-ray diffraction data collection by solving structures for three model proteins. The best resolution obtained in chip differed, compared with previously achieved resolutions, from significantly larger protein crystals and conventional X-ray data collection. This could be due to several factors and further crystallization condition optimization may further improve the diffraction. It was possible to collect in situ diffraction data up to 1.8 &#197; resolution applying crystal with dimensions smaller than 30 &#181;m. The detailed analysis of thaumatin diffraction data provided insights about radiation damage. To limit the extend of radiation damage, only one single crystal should be exposed per compartment in the microfluidic device, as the diffusion of radicals to and in neighboring crystals can occur. To improve the speed of data collection, this should be automated in future.
Due to crystal morphology, in some cases a preferred orientation can occur. This was for example the case with the thioredoxin dataset, where the crystals had a strongly preferred orientation relative to the chip windows. Even here, we could collect a complete diffraction dataset. If crystals exhibit a preferred orientation in chip and in particular if the corresponding space group has also a low symmetry, then the completeness of the dataset should be monitored during the collection such that sufficient diffraction patterns cane be collected.
Time-resolved studies using these chips are directly possible when using light induced reactions with a pump-probe approach. The polyimide foil light transmission for the pump laser needs to be elucidated and alternatively, optically clear polyimide or COC could be used. The current microfluidic geometries do not allow for substrate mixing experiments after the crystals are grown. However, we expect the described X-ray chip fabrication protocol to also be suitable for such mixing designs for both time-resolved X-ray diffraction as well as scattering approaches19.

Knowing the 3D structure of a protein is essential to understand its functionality. Near-atomic resolution structures are so far most commonly obtained by X-ray crystallography. This technique exposes protein crystals to X-ray radiation and the resulting diffraction patterns are then analyzed for structure determination and refinement. In traditional X-ray crystallography, a complete diffraction dataset is recorded from a single, ideally large, crystal at cryogenic temperatures. Such crystals, however, are mostly not trivial to grow, and to identify suitable cryo-preservation conditions can become challenging in itself and may sometimes also cause deviations from the native protein structure5.
Recent technological advances in X-ray free-electron laser (FEL) and synchrotron beamlines have allowed to solve structures from smaller crystals, as new micro-focusing beamlines, increased X-ray beam brilliance, and improved X-ray detectors became available6,7. Typically, small crystals are easier to grow than large and defect free crystals8,9. However, small crystals suffer from X-ray radiation damage much faster than large crystals. This is because compared to a large crystal, a higher X-ray dose must be projected into a smaller crystal volume to diffract to comparable resolution. Therefore, even cryogenic protection is often not sufficient to record a complete diffraction data set from a single microcrystal.
To overcome this hurdle, serial crystallography has become the method of choice to collect and merge diffraction patterns from many randomly oriented microcrystals to obtain a complete dataset. Radiation induced crystal damage is minimized by spreading the total X-ray dose used to solve a protein structure over a high number of crystals5,10. In a &#39;diffract before destroy&#39; FEL experiment, each crystal is only used for one exposure using femto-second X-ray pulses. Micro-focus beamlines at third generation synchrotron sources in turn can perform serial crystallography with a few millisecond short X-ray exposures11,12,13,14. Without a crystal oscillation or rotation during data collection, however, only partial Bragg reflections can be recorded and hence tens of thousands or more diffraction patterns are typically required for the structure determination15. To date, a diverse set of sample delivery methods has been developed for serial crystallography, as recently reviewed14,16,17,18,19. Amongst those, several fixed-target based sample delivery strategies were successfully combined with crystal rotation during X-ray exposures such that significantly fewer diffraction patterns can provide equally complete datasets while also consuming less sample compared to classical serial crystallography experiments where still images are recorded7,16,20,21,22,23,24.
We present a protocol to fabricate microfluidic devices with low X-ray background. The devices are patterned from 5-min epoxy glue using soft lithography and are suitable for in-situ X-ray diffraction experiments at room temperature that benefit from integrating the sample preparation directly into the X-ray set-up, as the case with time-resolved studies that follow mixing-induced kinetics18,19. Microfluidic channels are lidded on both sides with polymeric polyimide foil, resulting in X-ray windows with a combined thickness of about 16 &#181;m that allow for low X-ray background imaging. All used materials provide good solvent resistance. This fabrication method is comparably simple 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 setting.
In an application example, we describe chips for goniometer based fixed target serial crystallography. First, the design and fabrication considerations for using capillary valving to microfluidically split an aqueous reaction into a selected number of nanoliter sized droplets are discussed. This loading mechanism avoids the sample loss from channel dead-volume and splitting can easily be performed manually without using pumps or other equipment for fluid actuation. Such isolated nanoliter sized drops of protein solution are monitored in situ using dynamic light scattering (DLS) to control protein crystal nucleation and growth. It has previously been demonstrated that DLS measurements can be performed in microfluidic devices consisting of a polydimethylsiloxane (PDMS) structure bonded to a glass slide25,26. Because the polyimide layer has a high transmission for wavelengths longer than 550 nm, the approach can be extended to measurements in X-ray transparent chips as well, when using an appropriate laser wavelength27,28. Based on the DLS results, initial nucleation can be observed, and further droplet evaporation can be stopped to obtain fewer but larger protein crystals.
After sufficient crystals are grown, complete X-ray diffraction datasets can then be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. Diffraction datasets are processed using a suite of software tools and custom scripts to solve the protein crystal structure. This technique avoids artefacts often induced during cryo-preservation used in conventional crystallography experiments.
We compare three protein target structures that were solved using about 10-20 &#181;m small crystals grown in chip to better then 2 &#197; resolution. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. This protocol can be applied for protein crystals which diffract to high resolution as well as low resolution (1.7 &#197; to 3.0 &#197;). As almost every crystal can be used for diffraction, little sample is wasted, making this a very efficient crystal delivery method.
This protocol provides a detailed guide on how to prepare X-ray transparent microfluidic chips for in situ protein crystallization and diffraction data collection. The procedure was carefully designed to benefit from microfluidic precision without requiring sophisticated equipment in the lab. Also, data collection at the synchrotron beamline can be performed without requiring a specialized goniometer or humidifier to ease reproducing the results by non-experts. The presented technique can be applied for serial millisecond crystallography data collection at room-temperature while keeping the radiation damage minimal and without introducing stress to the crystals after growth by cryo-protection or crystal handling. Hence, the described method is suitable for any protein crystallization project.

<table><tbody><tr><td>SU-8 3000 Series</td><td>MicroChem Corp.</td><td>SU-8 3000</td><td>Photoresist</td></tr><tr><td>PGMEA</td><td>Sigma-Aldrich</td><td>484431</td><td>Developer</td></tr><tr><td>Isopropyl alcohol</td><td></td><td></td><td>Solvent</td></tr><tr><td>Ethanol</td><td></td><td></td><td>Solvent</td></tr><tr><td>Epoxy glue</td><td>UHU</td><td>Plus Schnellfest 5 min</td><td>Epoxy glue</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184</td><td>Silicone</td></tr><tr><td>Kapton foil</td><td>Dupont/ American Durafilm</td><td>HN grade, gauge 30 (7.5 &amp;mu;m)</td><td>polyimide foil</td></tr><tr><td>APTS</td><td>Sigma-Aldrich</td><td>440140</td><td>Chemical</td></tr><tr><td>GPTS</td><td>Sigma-Aldrich</td><td>440167</td><td>Chemical</td></tr><tr><td>Cytop CTX-109AE</td><td>Asahi Glass Co. Ltd</td><td>Cytop CTX-109AE</td><td>Cytop fluoropolymer coating</td></tr><tr><td>CT-Solv 100E</td><td>Asahi Glass Co. Ltd</td><td>CT-Solv 100E</td><td>Cytop fluoro-solvent</td></tr><tr><td>HFE-7500</td><td>3M</td><td>Novec 7500</td><td>Fluorinated oil</td></tr><tr><td>AutoCAD</td><td>AutoDesk Inc.</td><td>AutoCAD</td><td>CAD Software</td></tr><tr><td>Biopsy Punch</td><td>Harris</td><td>Uni-core 0.75 mm</td><td></td></tr><tr><td>Photo mask</td><td>JD Photo Data</td><td></td><td></td></tr><tr><td>3 inch wafer</td><td>University Wafer</td><td></td><td>Silicon wafer</td></tr><tr><td>Mask aligner</td><td>S&amp;Uuml;SS MicroTec</td><td>MJB4</td><td>Mask aligner</td></tr><tr><td>PDMS mixer</td><td>Thinky</td><td>ARE-250</td><td></td></tr><tr><td>Plasma machine</td><td>Diener electronic</td><td>Zepto</td><td></td></tr><tr><td>Thaumatin</td><td>Sigma Aldrich</td><td>T7638</td><td>Protein</td></tr><tr><td>Glucose Isomerase</td><td>Hamton Research</td><td>HR7-102</td><td>Protein</td></tr><tr><td>Bis-Tris</td><td>Sigma Aldrich</td><td>B9754</td><td>Chemical</td></tr><tr><td>Sodium Tartrate</td><td>Merck</td><td>106664</td><td>Chemical</td></tr><tr><td>Tris-HCl</td><td>Sigma Aldrich</td><td>10812846001</td><td>Chemical</td></tr><tr><td>HEPES</td><td>Carl Roth</td><td>6763.2</td><td>Chemical</td></tr><tr><td>Magnesium Chloride</td><td>Sigma Aldrich</td><td>208337</td><td>Chemical</td></tr><tr><td>Ammonium Sulfate</td><td>Sigma Aldrich</td><td>A4418</td><td>Chemical</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6758</td><td>Chemical</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>1064060250</td><td>Chemical</td></tr><tr><td>PEG1500</td><td>Molecular Dimensions</td><td>MD2-100-6</td><td>Chemical</td></tr><tr><td>SPG buffer</td><td>Jena Bioscience</td><td>CSS-389</td><td>Chemical</td></tr><tr><td>SpectroLight600</td><td>XtalConcepts</td><td></td><td>DLS Instrument</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td></td><td>Spectrophotometer</td></tr><tr><td>Zentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Ultimaker2</td><td>Ultimaker</td><td></td><td>3D printer</td></tr><tr><td>Form2</td><td>Formlabs</td><td></td><td>3D printer</td></tr><tr><td>Amicon Filter</td><td>Sartorius Stedim</td><td></td><td>0.2 &amp;micro;m filter</td></tr><tr><td>Tubing</td><td>Adtech Polymer Engineering Ltd</td><td>Bioblock/05</td><td>PTFE tubing 0.3 mm Inner Diameter x 0.76 mm Outer Diameter</td></tr><tr><td>Syringes&amp;nbsp;</td><td>BD</td><td>309628</td><td>1ml Luer-Lock Tip</td></tr><tr><td>Needle&amp;nbsp;</td><td>Terumo Agani Needle</td><td>AN*2716R1</td><td>27Gx5/8&quot;</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography at JoVE.com

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.

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 ...

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Research

JoVE Journal

Chemistry

14.6K Views.  DESY. 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.

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

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 ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Biochemistry

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 ...

High Pressure Single Crystal Diffraction at PX^2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the GSECARS 13-BM-C beamline at the Advanced Photon Source. The DAC program at 13-BM-C is part of the Partnership for Extreme Xtallography (PX^2) project. BX-90 type DACs with conical-type diamond anvils and backing plates are recommended for these experiments. The sample chamber should be loaded with noble gas to maintain a hydrostatic pressure environment. The sample is aligned to the rotation center of the diffraction goniometer. The MARCCD area detector is calibrated with a powder diffraction pattern from LaB6. The sample diffraction peaks are analyzed with the ATREX software program, and are then indexed with the RSV software program. RSV is used to refine the UB matrix of the single crystal, and with this information and the peak prediction function, more diffraction peaks can be located. Representative single crystal diffraction data from an omphacite (Ca0.51Na0.48)(Mg0.44Al0.44Fe2+0.14Fe3+0.02)Si2O6 sample were collected. Analysis of the data gave a monoclinic lattice with P2/n space group at 0.35 GPa, and the lattice parameters were found to be: a = 9.496 &#177;0.006 &#197;, b = 8.761 &#177;0.004 &#197;, c = 5.248 &#177;0.001 &#197;, &#946; = 105.06 &#177;0.03&#186;, &#945; = &#947; = 90&#186;.

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Bioengineering

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

Engineering

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

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.

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.

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

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

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

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