我们感谢CEMHTI（法国奥尔良，ANR-13-BS08-0012-01）和Labex OSUG@2020（法国格勒诺布尔，ANR-10-LABX-0056）为光束线开发提供的财政捐助。FAME-UHD项目由法国“grand emprunt”EquipEx（EcoX，ANR-10-EQPX-27-01），CEA-CNRS CRG财团和INSU CNRS研究所提供财政支持。我们感谢实验期间的所有贡献，特别是所有从事BM30B和BM16工作的人员。作者承认欧洲同步加速器辐射设施提供同步加速器辐射束时间。我们还感谢PHYTOMET ANR项目的财务支持（ANR-16-CE01-0008）和SEDMAC项目的财务支持（INCA-Plan cancer-ASC16019CS）。

1.制备用于硒形态的人类PC-3和OVCAR-3癌细胞颗粒
注：以下协议改编自Weekley等人10。所有步骤都必须在生物安全2级条件和限制下的细胞培养罩下使用无菌技术进行。
<ol><li>使用马拉塞兹细胞计数室对细胞进行计数。PC-3细胞系的每个烧瓶接种150，000-200，000个细胞，OVCAR-3细胞系接种300，000个细胞。</li>
	<li>T-75烧瓶中的种子细胞（每种条件下三个烧瓶，以便一式?...

<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>

该方案用于通过X射线吸收光谱研究生物样品中硒和铁的化学形式。它侧重于生物样品和参比化合物的冷冻制备和储存，以及HERFD-XAS的测量。
冷冻制备和储存 散装生物样品颗粒的冷冻制备可以保留样品中存在的物质的化学完整性。这是至关重要的，因为在使用冷冻干燥或空气干燥制备6时已经观察到形态变化。一旦选择用于测量的光束线配备了...

对必需或有毒元素的细胞生物转化的研究需要具有高灵敏度的物种形成技术，并应最大限度地减少通常容易改变化学物质的样品制备步骤。
已知硒和铁等生理元素特别难以形成，因为它们具有复杂的化学性质，硒或铁物种的各种稳定性，以及它们在ppm（mg / kg）甚至亚ppm范围内的低浓度。因此，研究XAS对这些元素的形态可能极具挑战性。同步加速器XAS和特别高能分辨率荧光检测的XAS（HERFD-XAS），允许非常低的信背景比1，可在同步加速器源上形成复杂生物基质中高度稀释的元素2，3。传统的荧光XAS测量可以使用能量带宽约为150-250 eV的能量分辨固态探测器（SSD）在欧洲同步辐射设施（ESRF）4的CRG-FAME光束线上进行，而HERFD-XAS测量则需要在ESRF2的CRG-FAME-UHD光束线上使用能量带宽约为1-3 eV的晶体分析仪光谱仪（CAS）.荧光光子分别通过电子或光学过程来区分其能量。
样品冷冻制备对于保存结构和维持成分化学完整性至关重要，因此允许分析接近生物天然状态5。此外，使用液氦低温冷却（LN2）在低至10 K的低温下进行的分析允许辐射损伤减缓并保持XAS的元素形态。虽然一些关于应用于生物样品的XAS技术的综述报告了在低温条件下制备和分析样品的必要性（例如Sarret等人6，Porcaro等人7），但没有一篇明确描述相关的详细方案。在本出版物中，描述了一种用于癌细胞和浮游生物微生物的冷冻制备方法，用于在低温下实现Se8 和Fe9 的HERFD-XAS形态。
在最先进的XAS光谱测量期间，样品制备和环境的良好实践需要1）设置;2）尽可能限制辐射损害影响的分析程序;3）样品（或模型化合物参考）相对于X射线光子光束尺寸尽可能均匀。通过使用液氦低温恒温器在低温下进行采集来考虑第一项。第二项是通过在样品的新区域上执行每次采集来处理的，方法是将其相对于光束移动。最后，考虑到第三个条件，样品（颗粒）和参比物（粉末）在压制的块状颗粒中进行调节，以尽可能限制孔隙率和不均匀性，并避免相对于X射线探测样品表面上的光束尺寸的粗糙度。我们解释了该协议如何处理所有这些要点。
我们利用人前列腺细胞系PC-3（高转移电位）和卵巢细胞系OVCAR-3（占所有卵巢癌病例的70%）来研究硒纳米颗粒（Se-NPs）和 Phaeodactylum tricornutum 硅藻对癌细胞的抗增殖特性，作为研究浮游植物铁螯合的模型物种。

<table border="0" cellpadding="0" cellspacing="0" style="width:2175px;" width="2174">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Ammonium nitrate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:389px;">A3795</td>
			<td style="width:1225px;">NH4NO3, 2.66 mg/L of milliQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anaerobic chamber</td>
			<td>Coy Laboratory, USA</td>
			<td></td>
			<td>equipped with Anaerobic Monitor (CAM-12)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Boron nitride powder</td>
			<td>Sigma-Aldrich</td>
			<td align="right">255475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell counting chamber</td>
			<td></td>
			<td></td>
			<td>Neubauer or Malassez</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell scraper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>GIBCO</td>
			<td>14190-094</td>
			<td>Without Calcium, Magnesium, Phenol Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf tubes</td>
			<td></td>
			<td></td>
			<td>0.5 mL and 1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon tubes</td>
			<td></td>
			<td></td>
			<td>15 mL and 50 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>A31604-02</td>
			<td>Performance Plus, certified One Shot format, US origin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z707503</td>
			<td>TPP 150 cm2 area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Growth chamber</td>
			<td>Sanyo</td>
			<td>Sanyo MLR-352</td>
			<td>at 20 &#176;C and under a 12:12 light (3,000 lux) dark regime</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>1 g/L of milliQ water HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td></td>
			<td></td>
			<td>Incubator at 37&#176;C, humidified atmosphere with 5% CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Manual hydraulic press</td>
			<td>Specac, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Morpholinepropanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M3183</td>
			<td>MOPS, 250 mg/L of milliQ water (pH 7.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC-3</td>
			<td>ECCAC, Salisbury, UK</td>
			<td align="right">90112714</td>
			<td>Storage temperature: liquid nitrogen vapor temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Pipette-boy</td>
			<td></td>
			<td></td>
			<td>25mL-, 10mL-, and 5mL sterile plastic-pipettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Plastic tweezers</td>
			<td>Oxford Instrument</td>
			<td>AGT 5230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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&#176;C in the dark, protected from the light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#176;C in the dark, protected from the light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium metasilicate pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">71746</td>
			<td>Na2SiO3.5H2O, 22.8 mg/L of milliQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium nitrate</td>
			<td>Sigma-Aldrich</td>
			<td>S5022</td>
			<td>NaNO3, 75 mg/L of milliQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5011</td>
			<td>NaH2PO4, 15 mg/L of milliQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-75 flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution (0.4%)</td>
			<td>GIBCO</td>
			<td align="right">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td>GIBCO</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Water bath 37&#176;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

在研究金属在生物系统中的作用时，用X射线吸收光谱（XAS）研究元素特别有趣。样品制备是一个关键且通常复杂的过程，特别是对于生物样品。虽然X射线形态形成技术被广泛使用，但尚未为该技术的用户传播详细的协议。此外，化学状态修饰是值得关注的，并且建议使用基于冷冻的技术来分析生物样品在其接近天然的水合状态下，以提供细胞或组织化学完整性的最大保存。在这里，我们提出了一种基于冷冻保存样品的细胞制备方案。它在高能分辨率荧光检测的X射线吸收光谱研究中证明了硒在癌细胞中的研究和浮游植物中铁的研究。该协议可与其他生物样品和其他可能被辐照破坏的X射线技术一起使用。

这些制剂的主要目的是研究硒纳米颗粒（Se-NPs）与癌细胞之间的相互作用，以及浮游植物中的铁结合和封存。
在初始状态（BSA Se-NPs）和在营养培养基中孵育的细胞（BSA Se-NPs）中孵育的硒的HERFD-XANES光谱如图 10所示。结果表明， 初始Se-NPs中的硒同时以Se（0） 和亚硒酸盐样形式存在， 而与PC-3细胞相互作用后， 细胞中的硒主要以Se（0）的形式<...

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

该协议提出了为基于同步加速器的X射线吸收光谱实验制备生物冷冻样品的详细程序。我们通过癌症和浮游植物细胞方案的示例描述了优化样品制备和冷冻保存所需的所有步骤。该方法提供了样品冷冻制备的通用标准。

使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析

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

该协议已针对细胞标本进行了标准化，允许比较在不同同步加速器位置运行的实验。该技术的主要优点是简单明了的工作流程，可以快速可靠地保存样品的化学整合。对这种技术不熟悉的个体可能会与冷冻的迭代细胞造粒和细胞沉淀快速加载到冷冻样品顺序中作斗争。在低温下精确快速地断开样品链接至关重要。因此，视觉演示对于了解如何执行该技术至关重要。为了制备用于硒形态的人前列腺和卵巢癌细胞系细胞沉淀，将每个细胞系中适当数量的细胞接种到每个条件下的三个T-75烧瓶中，在层流罩中的适当细胞培养基中，并将烧瓶放入37摄氏度和5%二氧化碳细胞培养箱中，直到细胞汇合80%。为了将癌细胞暴露于硒处理中，首先在室温下超声处理新鲜制备的纳米颗粒储备溶液在超声水浴中超声处理30分钟，然后将全细胞培养基中的硒纳米颗粒溶液连续稀释至适当的工作浓度。在层流罩中，每次洗涤用5毫升37摄氏度PBS轻轻洗涤细胞两次，并使用无菌的25毫升移液管小心地将15毫升感兴趣的硒处理液添加到烧瓶的底部。在不完全密封烧瓶的情况下关闭盖子，并将烧瓶水平放置在细胞培养箱中24小时。为了在洗涤和补充培养基后制备细胞沉淀，每个烧瓶使用一个细胞刮刀轻轻分离细胞。使用培养基将粘附在烧瓶上的任何细胞冲洗到上清液中，并通过离心收集解离的细胞。每次洗涤每管用五毫升PBS洗涤沉淀两次，以除去所有剩余的处理痕迹，并将细胞重悬于一毫升PBS中。将细胞转移到1.5毫升聚丙烯管中，并用另一次离心收集细胞。然后使用200微升移液管轻轻除去所有上清液，并将每个1.5毫升管的底部浸入液氮中至细胞沉淀的水平。冷冻后立即将管子转移到液氮杜瓦瓶中以长期储存。对于高分辨率X射线吸收光谱，在布拉格条件下，相对于感兴趣的荧光线能量优化晶体分析仪光谱仪的所有晶体，并使用已知吸收边缘能量位置的参考来校准入射单色束能量。将样品架转移到用于生物样品的液氦低温恒温器中，并将低温恒温器设置为10开尔文度，然后按照同步加速器安全规则关闭实验小屋，然后开始分析。在这里，具有代表性的高分辨率X射线吸收光谱光谱在初始状态下和在营养培养基中孵育的细胞中的硒表明，初始硒纳米颗粒中的硒以零硒和脂肪团样形式存在。然而，在与PC3细胞相互作用后，硒主要以零硒的形式存在于细胞中，这表明细胞内硒物种的变化。对于铁，高分辨率X射线吸收光谱参考光谱根据铁氧化态表现出不同的边缘位置，还原的铁种类转移到低能值。在硅藻颗粒的相同位置收集的两个连续光谱相似，表明当在10开尔文下使用氦低温恒温器时，两次采集之间的光束损伤受到限制。此外，硅藻颗粒不同位置的光谱相同，表明样品颗粒是均匀的，并且光谱可以平均以获得更好的信噪比频谱。最关键的步骤是纳米颗粒的制备，在低温下造粒，以及标准参考样品的制备。该程序可应用于其他本体分析技术，如ICP-MS或HPLC-ICP-MS或其他能够在低温下进行的非同步加速器光谱技术。使用这种方法可以探索其他科学问题，特别是在生物地质学，生物化学，环境科学或毒理学研究领域。硒化合物和硒纳米颗粒对您的健康可能同样艰巨，应该使用手套，眼镜和口罩在专用的化学罩中操纵。

Research

JoVE Journal

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Preparation of Carbon Nanosheets at Room Temperature

碳纳米片在室温下准备

Amphiphilic molecules equipped with a reactive, carbon-rich "oligoyne" segment consisting of conjugated carbon-carbon triple bonds self-assemble into ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Quantifying X-Ray Fluorescence Data Using MAPS

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Total Internal Reflection Absorption Spectroscopy (TIRAS) for the Detection of Solvated Electrons at a Plasma-liquid Interface

Total Internal Reflection Absorption Spectroscopy TIRAS for the Detection of Solvated Electrons at a Plasma-liquid Interface

全内反射吸收光谱法 (拉斯) 在等离子体-液体界面上检测溶剂电子

The total internal reflection absorption spectroscopy (TIRAS) method presented in this article uses an inexpensive diode laser to detect solvated ...

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

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

用于原位晶体 x 射线衍射和原位的微流控芯片系列晶体学的动态光散射

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

革兰氏阴性菌的基本金属摄取: x 射线荧光、放射性同位素和细胞分馏

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

软 x 射线吸收光谱和共振非弹性 x 射线散射法检测电池化学成分的元素敏感度

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

改进 x 射线激光时分辨序列飞秒晶体学微晶的高粘度挤出

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...