The experiments on the nano-imaging beamline ID16A were performed in the frame of ESRF proposals LS2430, LS2303, and LS2765.

<ol>
	<li>Lewis, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+synchrotron+nanoimaging+and+DNA+damage/genotoxicity+screening+of+novel+lanthanide-coated+nanovectors.">Intracellular synchrotron nanoimaging and DNA damage/genotoxicity screening of novel lanthanide-coated nanovectors.</a> Nanomedicine. 5 (10), 1547-1557 (2010).</li><li>Fus, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+localization+of+osmocenyl-tamoxifen+derivatives+in+hormone-independent+breast+cancer+cells+revealed+by+2D+and+3D+nano+X-ray+fluorescence+imaging.">The intracellular localization of osmocenyl-tamoxifen derivatives in hormone-independent breast cancer cells revealed by 2D and 3D nano X-ray fluorescence imaging.</a> Angwendte Chemie. , (2019).</li><li>Janssens, K., Adams, F., Rindby, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microscopic X-Ray Fluorescence Analysis. , (2000).</li><li>Carmona, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+exposure+of+human+dopaminergic+cells+results+in+low+cytotoxicity,+accumulation+within+sub-cytoplasmic+regions,+and+down+regulation+of+MAO-B.">Uranium exposure of human dopaminergic cells results in low cytotoxicity, accumulation within sub-cytoplasmic regions, and down regulation of MAO-B.</a> Neurotoxicology. 68, 177-188 (2018).</li><li>Leapman, R. D., Hunt, J. A., Buchanan, R. A., Andrews, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+low+calcium+concentrations+in+cryosectioned+cells+by+parallel-EELS+mapping.">Measurement of low calcium concentrations in cryosectioned cells by parallel-EELS mapping.</a> Ultramicroscopy. 49 (1-4), 225-234 (1993).</li><li>Saubermann, A. J., Echlin, P., Peters, P. D., Beeuwkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+scanning+electron+microscopy+to+X-ray+analysis+of+frozen+hydrated+sections.+I.+Specimen+handling+techniques.">Application of scanning electron microscopy to X-ray analysis of frozen hydrated sections. I. Specimen handling techniques.</a> Journal of Cell Biology. 88 (2), 257-267 (1981).</li><li>Saubermann, A. J., Heyman, R. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+concentration+of+high-energy+X-rays+for+diffraction-limited+imaging+resolution.">Efficient concentration of high-energy X-rays for diffraction-limited imaging resolution.</a> Optica. 4 (5), 492-495 (2017).</li><li>Studer, D., Humbel, B. M., Chiquet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+high-pressure+frozen+samples:+bridging+the+gap+between+cellular+ultrastructure+and+atomic+resolution.">Electron microscopy of high-pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution.</a> Histochemistry and Cell Biology. 130 (5), 877-889 (2008).</li><li>Moor, H., Steinbrecht, R. 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The authors have no conflicts of interest.

Cryo-electron microscopy (cryo-EM) won the 2017 Nobel Prize in chemistry and as such the development made by J. Dubochet on vitrification of biological material for the high-resolution structure determination of biomolecules in solution25. As reported by Dubochet in his Nobel lecture "Knowing how to vitrify a droplet of water is one thing, preparing a biological sample for biological observation is another"25. Cryopreparation steps are now considered the standard technique to mitigate radiation dose damage and study cells close to their native state. The preparation remains tedious, however. This is because electron microscopy, due to its unsurpassed spatial resolution, is sensitive to any ultrastructural artifact that occurs during the sample preparation. The synchrotron cryonanoprobes are now approaching similar difficulties going down to spatial resolutions as low as 13 nm in the high energy X-ray range26. Hard X-ray microscopy can analyze entire cells while electron microscopy suffers from the poor penetration depth of electrons enabling only very thin cell slices to be observed.
Monolayers of cells are thin enough so that by plunge-freezing in liquid ethane, the required cooling rates for water vitrification are attained. In theory, cooling rates as high as 108 K/s are possible using high-pressure freezing27 which allows vitrification of specimens too thick for plunge freezing. A cooling rate of 105 K/s, required to allow full vitrification of the sample at ambient pressure28, is reached reproducibly using the automatic plunge-freezing machine and parameters presented here. This allows a researcher to vitrify thin biological specimens (&#60;10 &#181;m) such as a monolayer of cells12,13,14,15,29,30 by plunge-freezing in liquid ethane.
An important challenge with this protocol is to also preserve as much as possible the chemical integrity of the intracellular content to provide reliable elemental distributions within the cell in 2D or 3D. As published elsewhere2,16,17,31, in the case of elemental imaging at the subcellular level, the analysis of frozen hydrated cells should be considered. Otherwise, the combination of plunge-freezing and freeze-drying of cells can be used for room temperature analysis. For the latter, the amorphous ice is removed through the process of sublimation, while the bound water molecules are removed through the process of desorption. This process may be far from ideal compared to frozen hydrated samples due to the possible alteration of the cellular membranes and the morphology of some subcellular structures32. Also, for speciation studies, the water extraction may lead to metal speciation artifacts. Still, it has been successful and the best alternative to frozen hydrated samples for elemental imaging at sub-100 nm levels2,16,17,18,20,33,34,35,36.
As it has been reported37, the quality of cryopreserved cellular preparations can be evaluated through the potassium-to-sodium K/Na ratio. Unfortunately, it cannot yet be determined with the hard X-ray nanoprobe used here, due to the low energy cut-off of the silicon drift detector used to detect the X-ray fluorescence photons of the elements (E &#8805; 1.3 keV magnesium). Indeed, a high K/Na ratio (&#62;10) that can be measured using TOF-SIMS, EPMA, or nuclear microprobe PIXE16,37 is indicative of the preserved chemical integrity of the cell compared to the expected K/Na of 25 in a living cell37. This can be supported by a concomitant low Cl/K ratio38. Still, imperfect vitrification, particularly if the speed of sample cooling is too low, can lead to the formation of large ice crystals that can damage cell membranes and organelles, consequently altering the distribution of chemical elements. Although there is no routine procedure to monitor this potential damage and impact on the intracellular distribution, the above elemental ratios and the possibility to image the cell at high resolution using X-ray phase contrast or cryo-soft X-ray microscopy can be the best approaches to support good preservation of intracellular compartments with concomitant preservation of the elemental integrity. The combination of these techniques and the use of newly developed cryocorrelative fluorescence optical microscopes will help assess to what extent this damage occurs and affects the intracellular elemental distribution.
Overall, a detailed and comprehensive protocol to prepare cellular samples for synchrotron X-ray fluorescence nano-analysis is presented. It is a good starting point for the research community, helping to solve the difficult issue of how to prepare appropriate cellular samples for 2D and 3D elemental imaging at (cryo) hard X-ray nanoprobes. These approaches can be merged with optical fluorescence and electron microscopy capabilities for in-depth correlative chemical and structural imaging of cells.

Newly designed synchrotron X-ray fluorescence (SR-XRF) nanoprobes allow visualization of the subcellular distribution of elements in a fully quantitative manner. As an example, this analytical capability allows investigation of the uptake of nanoparticles1 or organometallic molecules such as osmium-based complexes2, providing insight into the intracellular uptake of metal-based molecules with potent anticancer properties. As a multielement technique, SR-XRF3 with a nanoprobe provides a way to simultaneously quantify and localize intracellularly most biologically important elements, including phosphorus, sulfur, potassium, calcium, iron, copper, and zinc. Indeed, the use of hard X-rays provides large penetration depth to image whole frozen-hydrated cells in a label-free fashion. Furthermore, providing access to the K-edge of most elements of interest, the X-ray fluorescence is excited most efficiently. The use of cryogenic approaches allows reduction of radiation damage and optimization of the preservation of the cell structure and elemental distribution.
Most available spatially resolved analytical techniques to study metals in cells are surface techniques requiring very thin and flat sections of cells to be produced. This mainly encompasses scanning transmission electron microscopy with energy-dispersive X-ray analysis (STEM&#8211;EDX), energy-filtered transmission electron microscopy (EF-TEM), and nanoscale secondary ion mass spectrometry (nanoSIMS). The latter cannot be performed on frozen, hydrated cell sections while cryo-analysis can be done with electron microscopy with unsurpassed spatial resolution but poor elemental sensitivity. Particle-induced X-ray emission (PIXE) has allowed the study of elemental distributions in whole cells. It has the advantage of being fully quantitative with a fair elemental sensitivity at the micron scale and even at submicron resolution4, but suffers from radiation damage and lack of cryogenic capabilities to study frozen-hydrated cells. All these analytical techniques complement each another in the elemental imaging of cells, but for all techniques the sample preparation procedure is a crucial step. It should be kept simple to limit possible contamination as well as elemental redistribution and/or leakage to obtain meaningful results. As demonstrated in electron microscopy, a cryogenic workflow, including cryo-immobilization of the cell and cryotransfer to a cryoscanning stage, allows an optimal elemental preservation at subcellular levels as close as possible to the native state5,6,7,8,9,10. This understanding has been successfully implemented into the development of synchrotron cryo-soft X-ray microscopy (e.g., full field microscopes and scanning microscopes) to produce ultrastructural imaging of entire frozen-hydrated cells in 2D or 3D. Various cryogenic workflows were developed11 for soft X-ray microscopes at Beamline 2.1 (XM-2) of the Advanced Light Source at Lawrence Berkeley National Laboratory12, beamline U41-XM at the electron storage ring BESSY II (Germany)13, beamline MISTRAL of the ALBA light source (Spain)14, and at Beamline B24 of the Diamond light source15, among others. A similar workflow was recently shown to be the most reliable preparation and preservation method for intracellular elemental analysis using X-ray microprobes16,17.
Although X-ray nanoprobe techniques are starting to be widely used for cellular elemental analysis, particularly with the advent of cryogenic SR-XRF capabilities, no stepwise protocol has been disseminated so far to the research community. Here, a detailed procedure is provided to prepare cryofixed adherent cells cultured as monolayers on silicon nitride membranes to be analyzed under cryogenic conditions. A freeze-drying step to be applied after the protocol in case the X-ray analysis must be performed at room temperature is also provided. While the proposed protocol has been successfully used with human breast cancer cells MD-MB-2312 and the freeze-drying was demonstrated among others on mouse neurons18,20,21, it can be easily extended to various types of human or animal cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium Acetate solution, BioUltra, for molecular biology, ~5M in H2O</td>
			<td>SIGMA</td>
			<td>09691-250mL</td>
			<td>One can prepare the required solution from high-grade ammonium acetate powder and ultrapure water, pH and osmolarity needs to be adjusted anyway.</td>
		</tr>
		<tr>
			<td>B27 supplement, 50x</td>
			<td>Life Technologies, Invitrogen</td>
			<td>17504-044</td>
			<td>for hippocampal neuron culture</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate Buffered Saline, DPBS, ([-] CaCl2, [-] MgCl2)</td>
			<td>GIBCO</td>
			<td>14190-094</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>DMEM with Phenol Red/Glutamax I (Medium ATCC modification)</td>
			<td>GIBCO</td>
			<td>21885025</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle medium (DMEM)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>31966-02</td>
			<td>for hippocampal neuron culture</td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5, Straight Self-closing, 0.05x0.01mm Tips, Biology</td>
			<td>World Precision Instrument</td>
			<td>501202</td>
			<td></td>
		</tr>
		<tr>
			<td>Emitech K750X Peltier-Cooled EM Freeze Dryer</td>
			<td>Quorum Technology</td>
			<td>EK3147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethane N45</td>
			<td>Air Liquid</td>
			<td>p0505s05r0a001</td>
			<td>C2H6 &#62; 99,995 %</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Performance Plus, certified One Shot format, US origin</td>
			<td>GIBCO</td>
			<td>A31604-02</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>HBSS 10x</td>
			<td>Life Technologies, Invitrogen</td>
			<td>14185-052</td>
			<td>for hippocampal neuron culture</td>
		</tr>
		<tr>
			<td>Leica GP quick-release forceps</td>
			<td>Leica</td>
			<td>16706435</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 cell line, an epithelial, human adenocarcinoma breast cancer cell</td>
			<td>ATCC</td>
			<td>ATCC HTB-26</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies, Invitrogen</td>
			<td>21103-049</td>
			<td>for hippocampal neuron culture</td>
		</tr>
		<tr>
			<td>Nunc 4-Well Plate</td>
			<td>Thermo Fisher</td>
			<td>176740</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Osmo1 Single-Sample Micro-Osmometer</td>
			<td>Advanced Instruments</td>
			<td>Osmo1</td>
			<td>Alternative can be found at Fisher scientific (Wescor Inc. VAPRO&#174; Vapor Pressure Osmometer)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SIGMA</td>
			<td>P4333</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>SIGMA</td>
			<td>P4707</td>
			<td>Other type of coating can be used that is dependent of the cell type to be cultured on the membrane, other adhesion factors such as fibronectin, collagen, polyornithine can be tested accordingly. Cell can be cultured directly on silicon nitride membrane, but the latter are slightly hydrophobic and adhesion factors are recommended unless the membrane are processed to be hydrophilic (glow plasma discharged).</td>
		</tr>
		<tr>
			<td>Plunge freezing robot Leica EM GP main unit</td>
			<td>Leica</td>
			<td>16706401</td>
			<td>Alternative for automated plunger are the Vitrobot Mark IV (FEI), CryoPlunge 3 (Gatan), MS-002 Rapid Immersion Freezer (EMS). Manual home-made system can be used but an environment-controlled chamber is an asset for plunge-freezing.</td>
		</tr>
		<tr>
			<td>Silicon nitride membrane (Si3N4)</td>
			<td>Silson Ltd.</td>
			<td>SiRN-5.0(o)-200-1.5-500-NoHCl</td>
			<td>The proposed silicon nitride membrane type is optimised for analysis at ID16A ESRF X-ray nanoprobe, The 500 nm thickness of the membrane was chosen being more robust for cellular manipulation and cryofixation detailed within this protocol. Membrane with thickness of 200 nm or below can also be used although quite fragile, and other design of silicon nitride membrane can be purchased (for example TEM compatible membrane...) from Sislon or other company such as Norcada, SPI supplies, Ted Pella, EMS, LabTech, Neyco...</td>
		</tr>
		<tr>
			<td>Trypan blue solution 0.4%</td>
			<td>GIBCO</td>
			<td>15250061</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%</td>
			<td>GIBCO</td>
			<td>25300-054</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Ultratrace Elemental Analysis Grade, Ultrapure Water</td>
			<td>Fisher Chemicals</td>
			<td>W9-1</td>
			<td>MilliQ water can be used but has to be tested for trace element level of contamination using for example ICP-MS analysis.</td>
		</tr>
		<tr>
			<td>Whatman No. 1 filter paper with precut hole</td>
			<td>Leica</td>
			<td>16706440</td>
			<td>Alternative filter paper may be used and must have an outer diameter of 55 mm, the Punch for filter paper system from Leica (ref.16706443) can be used.</td>
		</tr>
	</tbody>
</table>

cell culture on silicon nitride membranes and cryopreparation for synchrotron x-ray fluorescence nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

A typical optical video microscope view of frozen hydrated MDA-MB-231 cells that were sub-cultured onto a poly-L-lysine coated Si3N4 membrane support is shown in Figure 7A. The optical view of the sample in the vacuum chamber was obtained in reflection mode using the dedicated online video microscope of the ID16A beamline of the ESRF22. While electron or soft X-ray microscopy requires the ice layer embedding the cell to be as thin as possible (typically &#60;0.5 &#181;m), hard X-rays (&#62;10 keV) have the advantage of a much higher penetration depth and lower dose deposition. The ice thickness can therefore be larger, typically &#60;10 &#181;m including the cell so that the ice embedding the cell is a few &#181;m in thickness. This can be estimated through the measured X-ray intensity in transmission compared to the intensity without the sample, taking into account the absorption of the 500 nm thick Si3N4 membrane. This ice thickness can be achieved through manual blotting as described in the present protocol. In the Newton rings region, the ice thickness can be even thinner (not measured).
The X-ray fluorescence elemental mapping of the frozen hydrated cell is shown in Figure 7B with the representative distributions of physiological elements such as potassium (K), sulfur (S), and zinc (Zn). These maps represent the elemental areal mass (i.e., elemental projected mass). While not done in the present case, such maps can be normalized through X-ray propagation-based phase contrast imaging that provides the estimation of the sample projected mass23. As reported by many studies, the highly diffusible K ion in cells preserved in their near-native state was assumed to be homogeneously distributed throughout the entire cell23,24,16. As shown in the 2D X-ray fluorescence elemental images in Figure 7B, the tightly bound element S was evenly distributed within the cell, similarly to K, and represents a good estimate of the cellular mass profile. The Zn distribution had a higher signal in the nucleus than in the cytosol and clearly outlined the nucleus. It can be noted that small Zn-enriched regions can be detected at the spatial resolution (50 nm) in the nuclear region.
The existing X-ray nanoprobes or the ones to be built do not necessarily accommodate cryogenic capabilities. In this case, the best alternative to get X-ray fluorescence images of cells at sub-100 nm spatial resolutions is to perform a freeze-drying procedure described in this protocol after plunge-freezing of the cell. Figure 8A shows a typical bright field microscopy view of resultant freeze-dried primary mouse hippocampal neurons directly cultured on the Si3N4 membrane. In this case, if stored in a clean desiccated chamber, the samples can be prepared 1&#8722;2 weeks in advance and be observed with an ordinary upright optical microscope for registration of regions of interest. Care should be taken to prevent exposure to ambient humidity as it may be captured by the freeze-dried sample and lead to damage under the X-ray nanobeam. This procedure was applied successfully to very sensitive cells (i.e., neuronal cells) and even better results were obtained with other more robust types of cells, such as cancer cells. As for plunge-frozen cells, the X-ray fluorescence images of K, S, and Zn on the entire freeze-dried cell display are similar to the ones described above. They are representative of the elemental distributions to be found in various types of freeze-dried cells at 50&#8722;100 nm spatial resolution. While freeze-drying whole cells is an alternative to preserve elemental integrity, it is at the expense of a perfect preservation of the cell morphology16, particularly cell membranes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60461/60461fig1.jpg" /> 
Figure 1: Typical sample support for X-ray fluorescence nano-analysis. A Si3N4 membrane support in its protective capsule. This type of substrate can be used both for room temperature analysis (plunge-freeze cellular preparation followed by low temperature and low vacuum freeze-drying process) or for cryogenic X-ray fluorescence analysis. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60461/60461fig02.jpg" /> 
Figure 2: Schematic view of the silicon nitride windows after cell seeding. The cells are cultured directly onto the poly-L-lysine coated flat surface of the Si3N4 membrane support. Sometimes air bubbles can be trapped in the backside cavity of the Si3N4 membrane support and have to be removed as described in the protocol. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60461/60461fig03.jpg" /> 
Figure 3: In-house developed 3D printed cryo-box for long-term storage of plunge frozen Si3N4 membrane supports in liquid nitrogen Dewar. (A) Cryo-box disassembled with the container and the caps (lower part) and (B) the assembled cryo-box with locked caps. The caps can be manipulated with the tweezers, opening or locking by rotation. A detailed plan for 3D printing is available upon request from ESRF ID16A. The design has been made to accommodate silicon nitride TEM grids. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60461/60461fig4.jpg" /> 
Figure 4: Blotting of cells cultured on Si3N4. Prior to plunge-freezing the cell monolayer cultured onto a Si3N4 membrane needs to be rinsed in ammonium acetate solution (A) and carefully manually blotted using filter paper (B). <a href="https://www.jove.com/files/ftp_upload/60461/60461fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60461/60461fig05.jpg" /> 
Figure 5: Automatic plunge-freezing EM-GP machine. (A) The automatic plunge freezer. (B) Environmental chamber with the tweezers locked in. (C) The ethane cup covered with the Leica liquefier connected to an ethane bottle. (D) The plunge-freezing enclosure showing the black cup full of liquified ethane and the cryo-box for further storage in LN2 of the vitrified Si3N4 membranes. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60461/60461fig06.jpg" /> 
Figure 6: Sample cryotransfer assembly for freeze-drying procedure. (A) The first brass recipient for Si3N4 membranes is mounted on top of the sample transfer holder provided by the freeze dryer supplier. (B) and (C) show that the second flat brass disc is used as a cover and acts as a cold trap enclosure to be inserted in the vacuum enclosure of the freeze dryer. (D) The full assembly with the spring-loaded transfer rod. (E) The sample holder carrying the vitrified cellular preparation grown on the Si3N4 membrane must be further inserted in the LN2-cooled freeze dryer. All the steps for mounting the assembly are done in LN2 in a Styrofoam box. For clarity, all the images were produced in the absence of LN2. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60461/60461fig07.jpg" /> 
Figure 7: Cryo-X-ray fluorescence images of a frozen hydrated cell using hard X-ray nanoprobe. (A) Typical online view in reflection mode using the dedicated optical video microscope of the ESRF ID16A beamline. After manual blotting, a total ice thickness of about 5&#8722;10 &#181;m was achieved that allows a clear view of the frozen hydrated cells. A region with Newton rings indicative of even much thinner ice is noticeable. (B) Representative cryo-X-ray fluorescence cellular distributions of physiological elements potassium (K), sulfur (S), and zinc (Zn). <a href="https://www.jove.com/files/ftp_upload/60461/60461fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60461/60461fig08.jpg" /> 
Figure 8: X-ray fluorescence images of a freeze-dried neuronal cell using hard X-ray nanoprobe. (A) Typical bright field microscopy view of resultant freeze-dried primary cortical neuronal cells directly cultured onto the Si3N4 membrane. Scale bar = 200 &#181;m (B) Representative room temperature X-ray fluorescence images of a single freeze-dried hippocampal neuron showing the distributions of physiological elements potassium (K), sulfur (S), and zinc (Zn). Scale bar = 2 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis at JoVE.com

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

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JoVE Journal

Biochemistry

9.0K Views.  Université Grenoble Alpes. Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

Video: Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

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

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

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

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

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

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. ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Fixed Target Serial Data Collection at Diamond Light Source

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.

We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is ...