Name: biological samples preparation for speciation at cryogenic temperature using high-resolution x-ray absorption spectroscopy
Uploaded: 2022-05-27T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution X-Ray Absorption Spectroscopy at JoVE.com

We are grateful for financial contributions to the beamline development by CEMHTI (Orleans, France, ANR-13-BS08-0012-01) and Labex OSUG@2020 (Grenoble, France, ANR-10-LABX-0056). The FAME-UHD project is financially supported by the French &#34;grand emprunt&#34; EquipEx (EcoX, ANR-10-EQPX-27-01), the CEA-CNRS CRG consortium and the INSU CNRS institute. We are grateful of all the contributions during the experiments especially all the persons working on BM30B and BM16. The authors acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation beamtime. We also acknowledge PHYTOMET ANR project for financial support (ANR-16-CE01-0008) and SEDMAC project for financial support (INCA-Plan cancer-ASC16019CS).

1. Preparation of the human PC-3 and OVCAR-3 cancer cell pellets for selenium speciation
NOTE: The following protocol is adapted from Weekley et al.10. All steps have to be carried out under a cell culture hood under biosafety level 2 conditions and restrictions, using aseptic techniques.
<ol>
	<li>Count the cells using a Malassez cell counting chamber. Seed 150,000&#8211;200,000 cells per flask for the PC-3 cell line and 300,000 cells for the OVCAR-3 cell line.</li>
	<li>Seed cells in T-75 flasks (three flasks per condition in order to have triplicates) in the adequate cell culture media (Table 1) under the laminar flow hood.</li>
	<li>Leave the cell cultures to grow in an incubator at 37 &#176;C and a humidified atmosphere with 5% CO2 until they reach 80% confluence. Usually, PC-3 prostate cancer cell lines double after 24 h while OVCAR-3 ovarian cancer cell lines take 72 h. The flasks must be left in the incubator.</li>
	<li>Meanwhile, dilute the Se-NPs used for treatment to a final concentration that corresponds to the IC20 (inhibitory concentration to achieve 20% cell death) that has been predetermined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for cytotoxicity assessment. These concentrations are specific to each Se-NPs type but also specific to the studied cell line.
	<ol>
		<li>Place the nanoparticle stock solution (2 mg/mL of&#160;bovine serum albumin [BSA] or chitosan coated Se-NPs) in an ultrasound water bath for 30 min at room temperature (RT).</li>
		<li>Dilute the Se-NPs solution by serial dilutions in complete cell culture medium to reach working concentrations. Between each dilution, vortex for 1 min.</li>
	</ol>
	</li>
	<li>Prepare a selenium positive control with diluted selenium salt for treatment.
	<ol>
		<li>Prepare an aqueous sodium selenite stock solution (1 mg/mL) in a 15 mL polypropylene tube. Mix 1 mg of sodium selenite powder with 1 mL of ultrapure water.</li>
		<li>Vortex the stock solution for 1 min.</li>
		<li>Dilute the sodium selenite stock solution by serial dilutions in complete cell culture medium to reach working concentrations. 
		NOTE: Do not forget to pipet several times before each dilution in order to homogenize the solution.</li>
	</ol>
	</li>
	<li>Expose the cancer cells to selenium treatments. 
	NOTE: This part of the protocol has to be repeated for each nanoparticle (NP) tested. Here, the NPs are of two types, BSA and chitosan coated Se-NPs, plus the controls. The sodium selenite solution is the positive control and no treatment is the negative control.
	<ol>
		<li>Open the three T-75 flasks containing the cells in the laminar flow hood.</li>
		<li>Remove the medium and wash the cells gently 2x with 5 mL of warmed (37 &#176;C) phosphate buffered saline (PBS).</li>
		<li>Gently, on the bottom-side of the flask and not directly on the cell layer, add 15 mL of the treatment in each flask using an automatic pipette equipped with 25 mL sterile plastic pipettes. Place the lids on the flasks. Do not close the lids tightly. 
		NOTE: As described in steps 1.3 and 1.4, treatment solutions must have been resuspended beforehand to be homogenized.</li>
		<li>Leave the three flasks horizontally in an incubator at 37 &#176;C and an atmosphere humidified with 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>Preparation of the cell pellets
	<ol>
		<li>Gently wash the cells 2x with 5 mL of warmed PBS (37 &#176;C) directly in the T75 flasks.</li>
		<li>Add 6 mL of warmed cell culture medium (37 &#176;C) on the cell layer in the T-75 flask using a pipette boy equipped with 10 mL sterile plastic pipettes.</li>
		<li>Collect the cells by gentle scraping using a cell scraper. Use one cell scraper per flask.</li>
		<li>Using a pipette boy and a 10 mL pipette, aspirate the liquid and flush back the cell/medium on the flask surface in order to collect all the cells. Repeat this step several times to dissociate and individualize the cells. Collect the medium containing the cells in a 15 mL polypropylene tube.</li>
		<li>Spin down at 250 x g for 5 min at RT. Aspirate the supernatant.</li>
		<li>Rinse the cells by resuspending them in 5 mL of PBS.</li>
		<li>Spin down at 250 x g for 5 min at RT. Aspirate the supernatant and repeat steps 1.6.5 and 1.6.6 2x to get rid of all remaining traces of the treatment.</li>
		<li>Resuspend the cells in 1 mL of PBS and transfer them into a 1.5 mL polypropylene tube. Finally, spin them down at 250 x g for 5 min at RT. Figure 1 represents the cell pellet obtained after centrifuging and discarding the supernatant.</li>
		<li>Gently remove all the PBS supernatant using a 200 &#181;L pipette. 
		NOTE: Be careful to not touch and damage the cell pellet. Ideally, the pellet should be 2&#8211;3 mm high or thick, with a diameter of 3&#8211;6 mm. For the PC-3 cell line, 9 x 106&#8211;10 x 106 cells are required for the assay. For the OVCAR-3 cell line, 8 x 106&#8211;9 x 106 cells are required (Figure 1). Some cells will likely be lost during the subsequent buffer rinsing steps. The higher the cell number, the better the pellet will be.</li>
	</ol>
	</li>
	<li>Flash-freezing of the cell pellets
	<ol>
		<li>Plunge the bottom part of the 1.5 mL tube containing the pellet in liquid nitrogen (LN2) to the level of the cell pellet.</li>
		<li>In a glove box fully purged with an inert atmosphere such as nitrogen gas, collect the cell pellet by gently tapping the 1.5 mL tube on the flat surface of a LN2-cooled copper block (Figure 2). The pellet is then collected without cell loss. 
		NOTE: Use cryoprotection equipment, lab coat, closed shoes, cryo-gloves, and a face shield or safety goggles for LN2 handling.</li>
		<li>If needed, a LN2 cooled needle can be used to help to take out the pellet. 
		NOTE: The resulting frozen cell pellet can fragment. These steps must be performed in the space of a few minutes and rely on the researcher&#39;s dexterity and speed.</li>
		<li>The frozen pellet dimensions must fit the cryostat sample holder. Using the equipment described, if the cell pellet is larger than 2 mm in height and slightly smaller than the hole of the sample holder used for XAS, the sample can be used directly. If not, or if a flat surface for the analysis is desired, a bulk cylindrical pellet must be prepared, still keeping the pellet in its frozen state. If the cell pellet is fragmented the following steps can be used ideally in a glove box fully purged with an inert atmosphere.</li>
	</ol>
	</li>
	<li>Preparation of the bulk frozen cylindrical pellets
	<ol>
		<li>Immerse all the parts of a manual hydraulic press that will be in contact with the sample in LN2 (Figure 3A; 3- or 5-mm diameter pellet dies, mold, piston/wire pulling).</li>
		<li>Transfer the rapidly frozen pellet fragments into the mold loaded with the appropriate pellet dies (Figure 3B).</li>
		<li>Quick pelletize with the hydraulic press. Use of 1.5 tons for a 5 mm diameter pellet or 0.5 tons for a 3 mm diameter pellet is sufficient (Figure 3C). 
		NOTE: After each pellet preparation with the hydraulic press, all the materials need to be thawed and dried properly using nitrogen gas to avoid any problems with the remaining frost and humidity.</li>
		<li>Collect the bulk pellet of frozen cells quickly and place it in an adequate cryotube for storage.</li>
		<li>Store it back into LN2 tank for long-term storage.</li>
		<li>Transfer and mount the cryo-pellets for analysis. The pellet stored in a LN2 cooled cryotube is transferred to a 77 K precooled cryostat sample holder in liquid N2 (Figure 4, as used for a CRG-FAME-UHD beamline). This step should be performed as quickly as possible but can take a few minutes. Then transfer the sample holder to the cryostat and maintain it at 10 K during the whole XAS analysis. 
		NOTE: Steps 1.9.3 and 1.9.6 are difficult because they need to be done quickly in order to avoid any frost formation that will block the piston and the mold. An alternative is to perform these steps under a plastic tent fully purged with nitrogen gas. The LN2-cool copper mass allows the pellet to be kept frozen.</li>
	</ol>
	</li>
</ol>
2. Preparation of the plankton cells for iron speciation
NOTE: Synthetic seawater used for this protocol was prepared by adding ultrapure water sea salts, morpholino propanesulfonic acid, ammonium nitrate, sodium nitrate, sodium metasilicate pentahydrate, sodium phosphate monobasic, vitamin stock, trace metal stock, antibiotic stock, and HEPES buffer at pH = 7.9. The concentrations of each component are indicated in the Table of Materials. Details on the culture can be found in Sutak et al.11
<ol>
	<li>Centrifuge a P. tricornutum diatom culture in a 50 mL polypropylene tube at 1,100 x g for 6 min and transfer the pellet into a flask with fresh medium.</li>
	<li>Let the culture grow in synthetic seawater in a growth chamber for 24 h.</li>
	<li>Centrifuge the plankton culture for 6 min at 1,100 x g and transfer the pellet into fresh medium containing Fe-citrate (1 &#181;M in the final culture). Incubate for 72 h.</li>
	<li>Prepare the plankton cells
	<ol>
		<li>Centrifuge the cells for 3 min at 1,100 x g and rinse with medium without iron.</li>
		<li>Transfer the pellet into a 1.5 mL polypropylene tube, wash with ultrapure water, and centrifuge 2x for 3 min at 1,100 x g. Remove the supernatant.</li>
		<li>Plunge the bottom part of the 1.5 mL polypropylene tube containing the pellet in LN2 as described in step 1.8.</li>
		<li>Transfer the frozen cells in a LN2 frozen molder and quickly press using a XRF pellet press (Figure 3 and Figure 7) as described in step 1.9.</li>
		<li>Remove the pellet and store it in a LN2 tank for long-term storage.</li>
		<li>Follow step 1.9.6 (see Figure 4 for the transfer into the cryostat sample holder).</li>
	</ol>
	</li>
</ol>
3. Reference compound preparation and measurement
NOTE: Reference compounds (solid or liquid) representative of the species and expected to be found in the biological system must be prepared and analyzed by XAS for comparison with experimental biological samples. Reference spectra can be also found in databases12,13,14 and can be used provided that the measurement conditions (e.g., spectral resolution) were similar to the experimental samples.
<ol>
	<li>For references in aqueous solution, weigh initial compounds in powder or liquid form under anaerobic condition or inert atmosphere. Prepare redox-sensitive references in an inert atmosphere (i.e., in an anaerobic glove box or in a Schlenk line, see Figure 5 and Figure 6) to avoid any oxido-reductive reaction and to preserve the chemical integrity of the compound, which can be highly reactive and unstable in ambient air). Here, all the selenium references were prepared using a Schlenk line and degasified ultrapure water (Figure 6). Liquid FeIII-malate and ferritine were prepared at ambient air.</li>
	<li>Mix with appropriate solution in order to reach a 1% wt final concentration of the studied element (i.e., selenium or iron) for collection in fluorescence mode. 
	NOTE: Ultrapure water is the most frequently used solution to prepare XAS references. Addition of glycerol (15%&#8211;20%) is required for liquids measured using standard XAS fluorescence to prevent artifacts arising from X-ray diffraction by ice crystals and preserved quality of the XAS signal collected with the solid-state detector.&#160;However, for HERFD-XAS, glycerol is not mandatory. Using a crystal analyzer spectrometer instead of a solid-state detector, the energy is selected with an extremely high-resolution and the diffraction induced by ice crystals does not impact the data collection of the studied element1.</li>
	<li>Preserve and store the prepared solution in a sealed Schlenk balloon (Figure 5) or in a 1.5 mL polypropylene tube until measurement in the same conditions as the samples.</li>
	<li>For solid references, weigh selenium or iron model compound powders at ambient air or in a glove box if the species are redox-sensitive. Here, solid FeII references (FeII-acetate and vivianite) were prepared in a gaseous N2 glove box while FeIII (Fe(OH)3, and FeIII-phosphate) were prepared at ambient air.</li>
	<li>Weigh high purity boron nitride powder and mix with the model compounds powders in order to reach a 1% wt final concentration of the studied element (Figure 7A).</li>
	<li>Grind to a fine powder using a mortar for at least 15 min.</li>
	<li>Place the powder mixture in the molder to prepare pellets with the hydraulic press (Figure 7B, similar to Figure 3A for the sample pellet).</li>
	<li>Close the molder with the piston (Figure 7C, similar to Figure 3B for the sample pellet).</li>
	<li>Press to obtain the bulk pellet (Figure 7D, similar to Figure 3C for the sample pellet).</li>
	<li>Store the prepared bulk pellets in the glove box until they are analyzed in the same conditions as the samples. 
	NOTE: The bulk pellet sometimes sticks to the pistons, depending on the powder compacity, for example. Removing it softly without breaking it then can be difficult. In that case, the following procedure using polyimide (e.g., Kapton) disks has to be used.</li>
	<li>Put a drop of ethanol on each side of the pistons that will be in contact with the powder (Figure 8A).</li>
	<li>Prepare two polyimide disks with a diameter slightly smaller than the pistons. The polyimide thickness needs to be 10&#8211;25 &#181;m (thinner will be difficult to manipulate, thicker will absorb too much of the X-ray photons during analysis).</li>
	<li>Put the disks on the pistons. They will stick by capillary action (Figure 8B).</li>
	<li>Mount the molder with the piston, add the powder, and put in the second piston (Figure 8C).</li>
	<li>Press (see step 3.11) and remove the compressed bulk pellet from the pistons. 
	NOTE: The solid references can be mounted on the cryostat-specific sample holder like the samples (see step 1.8.6). The liquid references can be mounted on the cryostat-specific sample holder. See Figure 9A for a CRG-FAME cryostat sample holder. The same procedure can be applied using a CRG-FAME-UHD sample holder, shown in Figure 4D, using the following procedure.</li>
	<li>Mounting of liquid reference on the cryostat sample holder.
	<ol>
		<li>Set the polyimide (e.g., Kapton) tape (25 &#181;m thick) in order to seal one side of the hole on the sample holder at RT (Figure 9B).</li>
		<li>Using a syringe, fill the cavity slowly with the solution until it reaches the top. Avoid bubbles. The reservoir must be filled (Figure 9C, left).</li>
		<li>Seal the other side of the cavity with the tape (Figure 9C, right).</li>
		<li>Plunge the sealed sample holder into LN2 (Figure 9D, T0&#8211;T3).</li>
		<li>A thermic reaction induces LN2 bubbling. Wait until bubbling stops, signaling the end of the reaction (Figure 9D, T3&#8211;T5).</li>
		<li>Transfer the sample holder at 77 K into the cryostat of the beamline before starting data collection (Figure 9D, T6) or storing in the LN2 Dewar. 
		NOTE: The frozen solution is well spread in the cavity and forms a uniform and homogeneous pellet (Figure 9D, T7).</li>
	</ol>
	</li>
</ol>
4. HERFD-XAS: measuring procedure
<ol>
	<li>Optimize all the crystals from the CAS in Bragg conditions with respect to the fluorescence line energy of interest. This procedure can be done with a concentrated reference compound.</li>
	<li>Calibrate the incident monochromatic beam energy using a reference for which the energy position of the absorption edge is known (typically a pure metallic foil). This reference can be positioned in a second step after the sample, in double transmission mode, in order to be able to check the calibration for each obtained spectra and eventually align them posttreatment.</li>
	<li>Position the sample in a liquid helium cryostat for biological samples following the appropriate procedure described in step 1.9.6.</li>
	<li>Close the experimental hutch following the synchrotron safety rules.</li>
	<li>Perform the XAS acquisition on an energy range that covers the selected absorption edge.</li>
	<li>After each spectral acquisition, move the sample at least twice the beam size in the direction of the motion before starting a new acquisition. 
	NOTE: In this case, measurements were performed using Ge(440) crystals to select the Fe Ka1 line and using Ge(844) crystals to select the Se Ka1 line. The beam size on the sample was 200 x 100 &#181;m&#178; (H x V Full Width Half Maximum). The sample motion between each acquisition was 500 &#181;m in both directions, in order to probe structural homogeneity and to analyze a new area free from previous possible radiation damage each time. Individual spectra are compared and merged if they are superimposable within the noise. The measurement is stopped for a given sample when the quality of the merged spectrum is correct. Then data analysis can be performed using any software dedicated to XAS analysis, especially those that allow a linear combination fitting of normalized spectra, for example Athena and Artemis (Demeter group of software)15, SixPack16, or Viper17. Complete and detailed explanations of XAS analysis falls outside the scope of this protocol and can be found in recent papers18,19, some of them more specifically devoted to biological samples20. Selenium XANES reference spectra are already gathered in the SSHADE spectra database21.</li>
</ol>

<ol>
	<li>Llorens, I., 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=High+energy+resolution+five-crystal+spectrometer+for+high+quality+fluorescence+and+absorption+measurements+on+an+x-ray+absorption+spectroscopy+beamline.">High energy resolution five-crystal spectrometer for high quality fluorescence and absorption measurements on an x-ray absorption spectroscopy beamline.</a> Review of Scientific Instruments. 83 (6), 063104 (2012).</li><li>Proux, O., 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=High+Energy+Resolution+Fluorescence+Detected+X-ray+Absorption+Spectroscopy:+a+new+powerful+structural+tool+in+environmental+biogeochemistry+sciences.">High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy: a new powerful structural tool in environmental biogeochemistry sciences.</a> Journal of Environmental Quality. 46 (6), 1146-1157 (2017).</li><li>Bissardon, 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=Sub-ppm+high+energy+resolution+fluorescence+detected+X-ray+absorption+spectroscopy+of+selenium+in+articular+cartilage.">Sub-ppm high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage.</a> Analyst. 144 (11), 3488-3493 (2019).</li><li>Proux, O., 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=FAME:+a+new+beamline+for+X-ray+absorption+investigations+of+very-diluted+systems+of+environmental,+material+and+biological+interests.">FAME: a new beamline for X-ray absorption investigations of very-diluted systems of environmental, material and biological interests.</a> Physica Scripta. 115, 970-973 (2005).</li><li>George, G. N., 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=X-ray-induced+photo-chemistry+and+X-ray+absorption+spectroscopy+of+biological+samples.">X-ray-induced photo-chemistry and X-ray absorption spectroscopy of biological samples.</a> Journal of Synchrotron Radiation. 19 (6), 875-886 (2012).</li><li>Sarret, G., 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=Use+of+Synchrotron-Based+techniques+to+Elucidate+Metal+Uptake+and+Metabolism+in+Plants.">Use of Synchrotron-Based techniques to Elucidate Metal Uptake and Metabolism in Plants.</a> Advanced in Agronomy. 119, 1-82 (2013).</li><li>Porcaro, F., Roudeau, S., Carmona, A., Ortega, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+element+speciation+analysis+of+biomedical+samples+using+synchrotron-based+techniques.">Advances in element speciation analysis of biomedical samples using synchrotron-based techniques.</a> Trends Analytical Chemistry. 104, 22-41 (2018).</li><li>Role of selenium nanoparticles to dampen the metastatic potential of aggressive cancer cells. 9th bioMedical Applications of Synchrotron Radiation, Beijing, China Available from: <a target="_blank" href="https://indico.ihep.ac.cn/event/7794/contribution/7">https://indico.ihep.ac.cn/event/7794/contribution/7</a> (2018)</li><li>Weekley, C. M., 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=Speciation+of+Seleno-amino+Acids+by+Human+Cancer+Cells:+X-ray+Absorption+and+Fluorescence+Methods.">Speciation of Seleno-amino Acids by Human Cancer Cells: X-ray Absorption and Fluorescence Methods.</a> Biochemistry. 50 (10), 1641-1650 (2011).</li><li>Sutak, R., 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=A+comparative+study+of+iron+uptake+mechanisms+in+marine+microalgae:+Iron+binding+at+the+cell+surface+is+a+critical+step.">A comparative study of iron uptake mechanisms in marine microalgae: Iron binding at the cell surface is a critical step.</a> Plant Physiology. 160, 2271-2284 (2012).</li><li>Asakura, K., Abe, H., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+constructing+an+international+XAFS+database.">The challenge of constructing an international XAFS database.</a> Journal of Synchrotron Radiation. 25 (4), 967-971 (2018).</li><li>SSHADE: "Solid Spectroscopy Hosting Architecture of Databases and Expertise" and its databases. OSUG Data Center. Service/Database Infrastructure Available from: <a target="_blank" href="https://www.sshade.eu/">https://www.sshade.eu/</a> (2018)</li><li>Bissardon, 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=Sub-ppm+high+energy+resolution+fluorescence+detected+X-ray+absorption+spectroscopy+of+selenium+in+articular+cartilage.">Sub-ppm high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage.</a> Analyst. 144 (11), 3488-3493 (2019).</li><li>Ravel, B., Newville, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATHENA,+ARTEMIS,+HEPHAESTUS:+data+analysis+for+X-ray+absorption+spectroscopy+using+IFEFFIT.">ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT.</a> Journal of Synchrotron Radiation. 12 (4), 537-541 (2005).</li><li>Webb, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIXpack:+a+graphical+user+interface+for+XAS+analysis+using+IFEFFIT.">SIXpack: a graphical user interface for XAS analysis using IFEFFIT.</a> Physica Scripta. 115, 1011 (2005).</li><li>Klementiev, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIPER+for+Windows.">VIPER for Windows.</a> Journal of Physics D: Applied Physics. 34 (2), 209-217 (2001).</li><li>Newville, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+of+XAFS.">Fundamental of XAFS.</a> Reviews in Mineralogy &amp; Geochemistry. 78, 33-74 (2014).</li><li>Henderson, G. S., de Groot, F. M. F., Moulton, B. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+Absorption+Near-Edge+Structure+(XANES)+Spectroscopy.">X-ray Absorption Near-Edge Structure (XANES) Spectroscopy.</a> Reviews in Mineralogy &amp; Geochemistry. 78, 75-138 (2014).</li><li>Ortega, R., Carmona, A., Llorens, I., Solari, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+absorption+spectroscopy+of+biological+samples.+A+tutorial.">X-ray absorption spectroscopy of biological samples. A tutorial.</a> Journal of Analytical Atomic Spectrometry. 27, 2054-2065 (2012).</li><li>Se K edge XAS HERFD of selenium with various oxidation states at 10K. SSHADE/FAME Available from: <a target="_blank" href="https://doi.org/10.26302/SSHADE/EXPERIMENT_CB_20190408_001">https://doi.org/10.26302/SSHADE/EXPERIMENT_CB_20190408_001</a> (2019)</li><li>George, G. N., 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=X-ray-induced+photo-chemistry+and+X-ray+absorption+spectroscopy+of+biological+samples.">X-ray-induced photo-chemistry and X-ray absorption spectroscopy of biological samples.</a> Journal of Synchrotron Radiation. 19, 875-886 (2012).</li></ol>

The authors have no conflicts of interest.

This protocol was used to study the chemical form of selenium and iron in biological samples by X-ray absorption spectroscopy. It focuses on the cryo-preparation and storage of biological samples and references compounds, as well as on the HERFD-XAS measurements.
Cryo-preparation and storage 
The cryo-preparation of the bulk biological sample pellets allows preservation of the chemical integrity of the species present in the samples. This is crucial, because speciation ch...

The study of the cellular biotransformations of essential or toxic elements requires speciation techniques with high sensitivity and should minimize sample preparation steps that are often prone to modification of chemical species.
Physiological elements such as selenium and iron are known to be particularly difficult to speciate due to their complex chemistry, various stabilities of the selenium or iron species, and their low concentration in the ppm (mg/kg) or even sub-ppm range. Thus, the study of the speciation of these elements by XAS can be extremely challenging. Synchrotron XAS and especially high energy resolution fluorescence detected XAS (HERFD-XAS), which allows a very low signal-to-background ratio1, are available at synchrotron sources to speciate highly diluted elements in complex biological matrices2,3. Conventional fluorescence-XAS measurements can be performed using an energy resolved solid state detector (SSD) with an energy bandwidth ~150&#8211;250 eV, on the CRG-FAME beamline at the European Synchrotron Radiation Facility (ESRF)4, while HERFD-XAS measurements need a crystal analyzer spectrometer (CAS), with an energy bandwidth ~1&#8211;3 eV, on the CRG-FAME-UHD beamline at the ESRF2. Fluorescence photons are discriminated with respect to their energy with electronic or optical processes respectively.
The sample cryo-preparation is essential to preserve structures and maintain compositional chemical integrity, thus allowing analysis close to the biological native state5. Moreover, analyses performed at cryogenic temperatures as low as 10 K using liquid helium cryogenic cooling (LN2), allow radiation damage to slow down and preserve elemental speciation for XAS. Although some reviews on XAS techniques applied to biological samples report the necessity to prepare and analyze samples in cryogenic conditions (e.g., Sarret et al.6, Porcaro et al.7), none of them clearly describes the related detailed protocol. In this publication, a method for cryo-preparation of cancer cells and plankton microorganisms is described for HERFD-XAS speciation of Se8 and Fe9 at cryogenic temperature.
Good practice for sample preparation and environment during state-of-the-art XAS spectroscopy measurements require 1) a setup; 2) an analysis procedure that limits the effects of radiation damage as much as possible; and 3) a sample (or model compound reference) as homogeneous as possible with respect to the X-ray photons beam size. The first item is taken into account by performing the acquisition at a low temperature, using a liquid helium cryostat. The second item is dealt with by performing each acquisition on a fresh area of the sample by moving it with respect to the beam. Finally, considering the third condition, samples (pellets) and references (powders) are conditioned in pressed bulk pellets in order to limit porosities and inhomogeneities as much as possible, and to avoid roughness with respect to the beam size on the X-ray probed sample surface. We explain how the protocol deals with all of these points.
We used human prostate cell line PC-3 (high metastatic potential) and ovarian cell line OVCAR-3 (which accounts for up to 70% of all ovarian cancer cases) to investigate the antiproliferative properties towards cancer cells of selenium nanoparticles (Se-NPs), and Phaeodactylum tricornutum diatom as a model species to investigate iron sequestration in phytoplankton.

<table><tbody><tr><td>Ammonium nitrate</td><td>Sigma-Aldrich</td><td>A3795</td><td>NH4NO3, 2.66 mg/L of milliQ water</td></tr><tr><td>Anaerobic chamber</td><td>Coy Laboratory, USA</td><td></td><td>equipped with Anaerobic Monitor (CAM-12)</td></tr><tr><td>Antibiotic stock</td><td>Sigma-Aldrich</td><td>A0166 for ampicillin, S9137 for streptomycin sulfate</td><td>1 mL/L of milliQ water (ampicillin sodium and streptomycin sulfate, 100 mg/mL)</td></tr><tr><td>Boron nitride powder</td><td>Sigma-Aldrich</td><td>255475</td><td></td></tr><tr><td>Cell counting chamber</td><td></td><td></td><td>Neubauer or Malassez</td></tr><tr><td>Cell scraper</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>GIBCO</td><td>14190-094</td><td>Without Calcium, Magnesium, Phenol Red</td></tr><tr><td>Eppendorf tubes</td><td></td><td></td><td>0.5 mL and 1.5 mL</td></tr><tr><td>Falcon tubes</td><td></td><td></td><td>15 mL and 50 mL</td></tr><tr><td>Ferric citrate Fe/citrate = 1/20</td><td>Sigma-Aldrich</td><td>F3388</td><td>aqueous solution of FeCl3 50 mM and Na-citrate 1M pH 6.5</td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td>A31604-02</td><td>Performance Plus, certified One Shot format, US origin</td></tr><tr><td>Flasks</td><td>Sigma-Aldrich</td><td>Z707503</td><td>TPP 150 cm2 area</td></tr><tr><td>Growth chamber</td><td>Sanyo</td><td>Sanyo MLR-352</td><td>at 20 &amp;deg;C and under a 12:12 light (3,000 lux) dark regime</td></tr><tr><td>HEPES buffer</td><td>Sigma-Aldrich</td><td>H4034</td><td>1 g/L of milliQ water HEPES</td></tr><tr><td>High grade serous, OVCAR-3</td><td>ATCC, Rockville, MD</td><td>HTB-161</td><td>Storage temperature: liquid nitrogen vapor temperature</td></tr><tr><td>Incubator</td><td></td><td></td><td>Incubator at 37&amp;deg;C, humidified atmosphere with 5% CO2</td></tr><tr><td>Insulin solution from bovine pancreas</td><td>Sigma-Aldrich</td><td>I0516</td><td>10 mg/mL insulin in 25mM HEPES, pH 8.2, BioReagent, sterile-filtered, suitable for cell culture</td></tr><tr><td>Manual hydraulic press</td><td>Specac, USA</td><td></td><td></td></tr><tr><td>Marine diatom Phaeodactylum tricornutum</td><td>Roscoff culture collection</td><td>RCC69</td><td>http://roscoff-culture-collection.org/rcc-strain-details/69</td></tr><tr><td>Morpholinepropanesulfonic acid</td><td>Sigma-Aldrich</td><td>M3183</td><td>MOPS, 250 mg/L of milliQ water (pH 7.3)</td></tr><tr><td>Optical microscope</td><td></td><td></td><td></td></tr><tr><td>PC-3</td><td>ECCAC, Salisbury, UK</td><td>90112714</td><td>Storage temperature: liquid nitrogen vapor temperature</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, sterile-filtered, BioReagent, suitable for cell culture</td></tr><tr><td>Pipette-boy</td><td></td><td></td><td>25mL-, 10mL-, and 5mL sterile plastic-pipettes</td></tr><tr><td>Plankton culture products, Mf medium: Sea salts</td><td>Sigma-Aldrich</td><td>S9883</td><td>40g/L of milliQ water. Composition: Cl- 19.29 g, Na+ 10.78 g, SO42- 2.66 g, Mg2+ 1.32 g, K+ 420 mg, Ca2+ 400 mg, CO32- /HCO3- 200 mg, Sr2+ 8.8 mg, BO2- 5.6 mg, Br- 56 mg, I- 0.24 mg, Li+ 0.3 mg, F- 1 mg</td></tr><tr><td>Plastic tweezers</td><td>Oxford Instrument</td><td>AGT 5230</td><td></td></tr><tr><td>RPMI MEDIUM 1640 (ATCC Modification)</td><td>GIBCO</td><td>A10491-01</td><td>Solution with 4.5 g/L D-glucose, 1.5 g/L Sodium Bicarbonate, 110 mg/L (1 mM) Sodium Pyruvate, 2.388 g/L (10 mM) HEPES buffer and 300 mg/L L-glutamine for research use</td></tr><tr><td>Selenium nanoparticles (Se-NPs), BSA coated, 2 mg/mL</td><td>NANOCS Company, USA</td><td>Se50-BS-1</td><td>BSA stabilized Se-NPs solution. Average size about 30 nm. Stored at 4&amp;deg;C in the dark, protected from the light.</td></tr><tr><td>Selenium nanoparticles (Se-NPs), Chitosan coated, 2 mg/mL</td><td>NANOCS Company, USA</td><td>11. Se50-CS-1</td><td>Chitosan stabilized Se-NPs solution. Average size about 30 nm. Stored at 4&amp;deg;C in the dark, protected from the light.</td></tr><tr><td>Sodium metasilicate pentahydrate</td><td>Sigma-Aldrich</td><td>71746</td><td>Na2SiO3.5H2O, 22.8 mg/L of milliQ water</td></tr><tr><td>Sodium nitrate</td><td>Sigma-Aldrich</td><td>S5022</td><td>NaNO3, 75 mg/L of milliQ water</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S5011</td><td>NaH2PO4, 15 mg/L of milliQ water</td></tr><tr><td>T-75 flasks</td><td></td><td></td><td></td></tr><tr><td>Tissue culture hood</td><td></td><td></td><td></td></tr><tr><td>Trace metal stock</td><td>Sigma-Aldrich</td><td>M5005, Z1001, M1651, C2911, 450243, 451193, 229857</td><td>1 mL/L of milliQ water (MnCl2.4H2O 200 mg/L, ZnSO4.7H2O 40 mg/L, Na2MoO4.2H2O 20mg/L, CoCl2.6H2O 14 mg/L, Na3VO4.nH2O 10 mg/L, NiCl2 10 mg/L, H2SeO3 10 mg/L)</td></tr><tr><td>Trypan Blue Solution (0.4%)</td><td>GIBCO</td><td>15250061</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>GIBCO</td><td>25300-054</td><td></td></tr><tr><td>Vitamin stock</td><td>Sigma-Aldrich</td><td>T1270 for thiamine, B4639 for biotin, V6629 for B12</td><td>1 mL/L of milliQ water (thiamine HCl 20 mg/L, biotin 1 mg/L, B12 1 mg/L)</td></tr><tr><td>Water bath 37&amp;deg;C</td><td></td><td></td><td></td></tr></tbody></table>

biological samples preparation for speciation at cryogenic temperature using high-resolution  x-ray absorption spectroscopy

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

The main aims of these preparations were to investigate the interaction between selenium nanoparticles (Se-NPs) and cancer cells, and iron binding and sequestration in phytoplankton.
HERFD-XANES spectra of the selenium in the initial state (BSA Se-NPs) and in cells incubated in nutritive medium (BSA Se-NPs after 24 h incubation) are shown in Figure 10. Results showed that selenium in the initial Se-NPs was present as both Se(0) and selenite-like forms, ...

Watch this Scientific Journal Video about Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy at JoVE.com

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

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

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

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Research

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Chemistry

2.5K Views.  INSERM UA7, Synchrotron Radiation for Biomedicine (STROBE). Dit protocol is gestandaardiseerd voor cellulaire monsters, waardoor experimenten die op verschillende synchrotronlocaties worden uitgevoerd, kunnen worden vergeleken. Het belangrijkste voordeel van deze techniek is een eenvoudige en ongecompliceerde workflow die een snelle en betrouwbare conservering van de chemische integratie van het monster mogelijk maakt. Personen die nieuw zijn in deze techniek kunnen worstelen met de pelletering van bevroren iteratieve cellen en het snel laden van de c...

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

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Environment

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.

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 ...

Biochemistry

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

Biology

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

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

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