Name: Mikrosıvısal çip Situ Crystal x-ışını kırınım ve in Situ dinamik ışık saçılma seri kristalografisi için için (Video) | JoVE Journal
Uploaded: 2018-04-24T13:00:00.000+00:00
Duration: 11 min 48 s
Description: Öğrenin, mikrosıvısal çip kullanarak oda sıcaklığında protein kristalleştirme ve X-ışını kırınım verisi toplama yöntemini. Dinamik ışık saçılması ile kristalleşme sürecini izleyin ve yapısal analiz yapın.

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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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</ol>

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

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.

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 ...

The overall goal of this procedure is to fabricate and operate a microfluidic device for protein crystallization X-ray diffraction data collection at room temperature. The main advantage of this technique is that crystallization inside the chip minimizes the mechanical disturbance of the protein crystals. These X-ray transparent chips are easy to produce, can be directly mounted on goniometers of most synchrotron beamlines, and make efficient use of available crystals when collecting X-ray diffraction data.First, place a master mold of the X-ray chip design in a 10-centimeter Petri dish lined with aluminum foil. Mix together about 30 grams total of PDMS base and curing agent in a 10-to-one ratio, and pour the PDMS onto the master to a height of four millimeters. Degas the PDMS in a vacuum desiccator for five minutes, and blow air to remove remaining air bubbles on the surface of the PDMS.Cure the PDMS in an oven at 70 degrees Celsius for one hour. Then, gently peel the cured PDMS mold from the master. Use a scalpel to cut away the excess PDMS.Prior to X-ray chip fabrication, ensure that the workspace is arranged to allow easy access to all needed equipment and components during epoxy mold casting. To begin the chip fabrication, dilute the precursors of a two-component epoxy resin with ethanol to a final ethanol mass concentration of 40%by weight. Using a vortex mixer, mix epoxy resin and ethanol together.Degas the PDMS mold in a vacuum desiccator for 30 minutes to ensure that the PDMS can later absorb small air bubbles from the epoxy resin. Cut a 70 millimeter by 70 millimeter piece of 7.5-micron thick polyimide foil, and wrap the foil around a 75 millimeter by 50 millimeter glass slide. Tape the edges of the foil to the back of the slide to form a flat, rigid polyimide surface.Treat the slide-backed polyimide foil with oxygen plasma for 20 seconds. Then, incubate the foil in a 1%by volume aqueous solution of either APTS or GPTS for five minutes at 20 degrees Celsius. During silanization, thoroughly mix together the epoxy resin precursor solutions with a small spatula.Transfer the degassed PDMS mold from the vacuum desiccator to a flat surface. Dry the silanized polyimide foil with pressurized air or nitrogen gas. Quickly apply a droplet of the resin mixture to each microstructure on the mold.Place the slide-backed polyimide foil face down on the epoxy resin, and firmly press down on the slide. Cover the assembly with a plastic foil and a metal sheet. Place weights on the metal sheet to apply a pressure of up to 1.4 newtons per square centimeter to the assembly.After 15 minutes, gently separate the glass slide from the epoxy-patterned polyimide foil, and wipe the polyimide foil until dry. Place plastic foil, metal sheet, and weights back on top. Allow the epoxy to cure at room temperature for one hour.Afterwards, remove the weights, the metal sheet, and the plastic foil. Gently peel the patterned polyimide foil from the PDMS mold. Plasma treat the patterned foil for 20 seconds, and silanize the patterned foil with either APTS or GPTS, as previously described.Silanize a pristine polyimide foil with the other silane solution under the same conditions. Dry both foils with pressurized air. Then, apply a drop of deionized water to a clean, dry, flat surface.Cut individual chip structures on polyimide foil, and place the patterned polyimide foil epoxy side up on the drop of water, and carefully smooth out the foil until completely flat. Place the second polyimide foil on the patterned foil with the silanized face down. Gently hand smooth the second foil from corner to opposite corner to expel air bubbles and to bond the polyimide sheets together.To begin fabricating the access ports, cut from a cured, four-millimeter PDMS slab a block of sufficient size to cover all ports in the polyimide X-ray chip without covering the crystallization compartment. Plasma treat, silanize, and dry the polyimide chip and the PDMS block. Press the treated block onto the chip over the ports.Cover the chip with a plastic foil, a clean glass slide, and a metal sheet. Apply 1.4 newtons per square centimeter of pressure for one hour. Next, use a 0.75-millimeter biopsy punch to punch inlet and outlet holes where marked in the chip design.Seal the back of the chip with polyimide film electrical insulating tape. Then, dilute a 9%by weight stock solution of transparent fluoropolymer coating material to 0.45%by weight with a fluorinated solvent. Connect a 27-gauge, 5/8-inch needle to the syringe.Fit polytetrafluoroethylene tubing over the end of the needle. Load the fluoropolymer solution into a one-milliliter Luer lock syringe. Connect the other end of the tubing to the X-ray chip outlet.Inject the fluoropolymer solution into the chip until all channels are filled, and remove excessive fluoropolymer solution by using a syringe filled with air. Place the filled chip with the flat side down on a hotplate at 190 degrees Celsius. Heat the chip for five minutes to evaporate the solvent, thereby coating the channels with the fluoropolymer.Prior to the crystallization procedure, filter the chosen protein solution with a 0.2-micron filter. Centrifuge the solution for 15 minutes at 16, 100 times g and 20 degrees Celsius, and recover the supernatant for the crystallization experiments. To begin the crystallization procedure, combine equal volumes of a protein solution and its precipitant.Draw about 20 microliters of this mixture into a syringe. Use a 27-gauge, 5/8-inch needle and PTFE tubing to connect the syringe to the inlet of the X-ray chip. Connect a syringe of fluorinated oil to the outlet in the same way.While monitoring the chip under a microscope, fill the crystallization solution through the inlet into the chip with the parallel layout. Inject sufficient fluorinated oil to separate the crystallization compartments in the chip. Then, block both the inlet and outlet ports with paperclips or other narrow plugs.Store the chip under the appropriate crystallization conditions. The crystal formation can now be observed under a microscope or by dynamic light scattering measurements. When ready for data collection, use double-sided tape to fix the X-ray chip to a 3D-printed adapter for the plate goniometer.Mount the chip adapter on the plate goniometer at the synchrotron beamline. Collect and analyze the diffraction data. Bright field microscopy was used to quantify the evaporation rate of water from the crystallization chambers of the polyimide foil chip.Dynamic light scattering could detect the initial nucleation of thaumatin crystals in a PDMS chip on a glass slide with equivalent geometry, allowing crystallization conditions to be optimized. 10 diffraction patterns were collected from each of 83 thaumatin crystals grown in a polyimide chip with a one-degree rotation during each frame. The dataset was split into five sub-datasets by frame, and the intensity decay of the normalized diffraction power over time was then evaluated.By the fourth dataset, the diffraction power had decreased to below 50%The measurement R factor values of the sub-datasets increased over time, indicating that the crystals sustained radiation damage during data collection. This was attributed to free radicals generated during X-ray exposure degrading neighboring crystals in the same compartment. The bipyramidal thaumatin crystals had a broad distribution of orientations in the X-ray chip.Orthorhombic glucose isomerase crystals likewise showed a broad distribution of orientations when grown in a polyimide chip. However, thioredoxin crystals predominantly had orientations in the xy, xz, and yz planes, potentially owing to their elongated shape. In general, these chips are very robust and have a low X-ray background.This makes this fabrication method suitable for other X-ray imaging techniques, such as small-angle X-ray scattering. This procedure can be easily adapted to suit your experimental needs. For instance, the chip design can be modified by adjusting the dimensions of the individual compartments or the total number of compartments per chip.A particular focus for us was to engineer a workflow that does not require cost-intensive equipment, like specialized tools or microfluidic pumps. After watching this video and reading the manuscript, you should have a good understanding how to crystallize in chip, to monitor the crystallization process, and to record and analyze diffraction data using the chip approach. Once mastered, this technique enables batch fabrication of microfluidic chips from a PDMS master in about three hours without requiring a dedicated cleanroom.

microfluidic-chips-for-situ-crystal-x-ray-diffraction-situ-dynamic

Research

JoVE Journal

Chemistry

Kimya

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Burada, mikrokristal elektron kırınımı (MicroED) deneyleri için küçük moleküllü kristallerin tozlarının hazırlanması için laboratuvarımızda geliştirilen prosedürleri açıklıyoruz.

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Burada, EMBL Grenoble'daki HTX Laboratuvarı'nda KristalDirekt teknolojisine dayalı proteinden yapıya, hızlı ligand-protein kompleksi analizi ve büyük ölçekli parça taraması için otomatik makromoleküler kristalografi boru hatlarının nasıl kullanılacağını açıklıyoruz.

Grenoble'daki EMBL HTX Tesisinde Otomatik Kristalografi Boru Hatları

Bu katkı, kristal üzerinde kristal cihazlarda protein kristalizasyonunun nasıl kurulacağını ve çip üzerinde kristalizasyon platformunu kullanarak oda sıcaklığında otomatik seri veri toplamanın nasıl gerçekleştirileceğini açıklamaktadır.

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

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