化学固定准备贴壁细胞的X射线荧光成像
Summary
Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.
Abstract
X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.
Introduction
X射线荧光成像可以用于两者的身份和样品中存在的元素的量，以在空间上分辨。选择为大于电子结合感兴趣的重元素的能量的能量的入射X射线，克服内壳层电子的结合能的核1。这会在电子层一个“洞”。作为高能量电子落入到这些孔中，荧光X射线被发射的，其波长依赖于那些轨道的能量的分离。由于轨道的能量间距是一个给定的元件的特性，在X射线荧光发射也具有特征波长，取决于在元件上。正是这种发射在特征波长，允许存在的元素的识别。的荧光强度的校准允许存在的元素的定量分析。
X射线荧光microscopY（XFM）已经越来越使用，部分原因是非常辉煌的X射线同步辐射光源，例如那些在春天-8在日本，欧洲同步加速器辐射设备（ESRF）在法国，以及先进光子源（发展APS），在美国的2。这些源提供非常高强度X射线束。同时，改善在透视光学，如带片的技术，允许这些光束到亚微米的斑点的聚焦，虽然相当低效3。具有非常高强度的光束，即使是相对小的光量，可以集中足以激发内源的金属中的细胞，产生的信号，可以与目前可用的检测器技术来测量。因此，研究金属的化学生物学中，细胞是在特定的一个应用程序，利用许多的最近发展的在该技术4-10。
存在要考虑的，而应用程序的许多关键因素卧XFM调查培养的哺乳动物细胞或其它生物样品的元素分布和定量。首先，将样品需要保持完好，在结构上并且相对于它的元素组成中，为了进行测定才有意义。其次，样品也必须以某种方式保存，使得它是耐寒到能够通过聚焦X射线束而引起的辐射损伤。该样品可以一次同时满足这些标准的一种方法是将快速冷冻成玻璃状，无定形冰11,12。快速冷冻往往是通过诸如暴跌冻结或高压冻结13-16各种冷冻保存技术来实现的。人们普遍认为冻存保留整体蜂窝结构，化学成分的生物样品尽可能接近天然状态为可能的。化学固定，另一方面，由于缓慢和选择性渗透固定剂进入细胞和组织如瓦特ELL在膜渗透性作为以后的变化，可以允许各种细胞的离子特别是可扩散的离子如Cl，Ca和K至浸出，丢失或重定位，从而使这些元件不理想的17-19的调查。尽管低温定影超过化学固定在一般情况下，对于贴壁哺乳动物细胞，尤其是明显的优势，冷冻保存有各种限制20-23。其中最明显的是，并不是每一个研究实验室可方便前往冷冻保存仪器。大多数当前的高压冷冻甚至沉浸冷冻费用昂贵，拥有仅由冷冻设施的子集，其可以远离其中细胞温育。冷冻保存的好处可能被交易的出行压力置于细胞的缺点。因此，尽管冻存肯定是最严格的方式来保存样品X射线荧光分析，它肯定不是最方便的在各种情况下所有的研究人员;也不总是必要 – 如果感兴趣的金属是紧密地结合到可定影大分子，并且在该样品将被成像的分辨率大于在干燥过程中可能出现的超显微组织的损害。注意到的告诫24的，化学固定和干燥可以是合适的选择。
其他因素在成功的X射线荧光成像实验，包括适当的分析。 X射线荧光成像是从根本上的X射线荧光发射光谱结合光栅扫描，以提供空间分辨率。收集到的X射线荧光发射光谱重叠的包含发射峰，背景和入射光束的弹性和非弹性散射峰的组合。软件，使反卷积的这些贡献和排放峰值的拟合，得到了关键的发展，这一领域的25。此外，开发和商业DIST已知成分，用于校准相对于物质量荧光强度薄膜标准ribution，也一直很重要。
这个协议提供了通过化学固定和空气干燥制备贴壁细胞的描述。在这个过程中的重要一步是细胞上的氮化硅窗口，往往不坚持好，使温柔漂洗以特殊的方式成功的关键的增长。
Protocol
Representative Results
Discussion
在许多领域，包括地球科学，材料科学，化学生物学26-34 X射线荧光成像是非常有用的。进展同步辐射X射线，和它们的聚焦，产生了非常高的强度的光束。聚焦X射线束足以激发内源的金属中的细胞现在存在，产生可与目前可用的硅漂移检测器技术进行测量的信号。和学习金属的化学生物学中，细胞是在特定的一个应用程序，利用许多近期的发展在这方面的。
然而，使…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge Stefan Vogt for his assistance in the fitting of the representative data shown in this paper, and helpful discussions. The authors also acknowledge Chris Jacobsen for his support to Q. J.
Use of the Advanced Photon Source, beamlines 2-ID-E and 8-BM-B, at Argonne National Laboratory was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.
Materials
| silicon nitride windows | Silson Ltd/J B J Business Park/Northampton Rd, Northampton NN7 3DW, United Kingdom | No part numbers available. Order by size. Membrane size: 1.5 mm x 1.5 mm. Thickness 500 nm. Frame size: 5 mm x 5 mm. Frame thickness: 200 µm | Alternate source: SPI Supplies / Structure Probe, Inc.West Chester, PA |
| reverse tweezers | Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450 | 78520-5X | EMS 5X, NC – Ultra Fine Tweezers |
| rubber grid mat | Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450 | 71170 | Round Grid Mat |
| acetic acid | Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052 | 338826 | trace metals grade concentrated acetic acid |
| PIPES buffer | Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052 | P6757 | solid PIPES buffer |
| formaldehyde stock solution | Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450 | RT 17113 | 10 x 10mL ampules of 20% aqueous paraformaldehyde |
References
- Thompson, A. C. . Center for X-ray Optics and Advanced Light Source. , 1-53 (2009).
- Helliwell, J. R. Synchrotron radiation facilities. Nat. Struct. Biol. 5, 614-617 (1988).
- Lai, B., et al. X-ray Phase Zone Plate Fabricated by Lithographic Techniques. Appl. Phys. Lett. 61 (16), 1877-1879 (1992).
- Dodani, S. C., et al. Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy. Proc. Natl. Acad. Sci. U. S. A. 108 (15), 5980-5985 (2011).
- Finney, L., et al. X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 104 (7), 2247-2252 (2007).
- Kehr, S., et al. X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis. J. Mol. Biol. 389 (5), 808-818 (2009).
- McCormick, N., Velasquez, V., Finney, L., Vogt, S., Kelleher, S. L. X-ray fluorescence microscopy reveals accumulation and secretion of discrete intracellular zinc pools in the lactating mouse mammary gland. PloS One. 5 (6), (2010).
- Paunesku, T., Vogt, S., Maser, J., Lai, B., Woloschak, G. X-ray fluorescence microprobe imaging in biology and medicine. J. Cell. Biochem. 99 (6), 1489-1502 (2006).
- Twining, B. S., et al. Quantifying trace elements in individual aquatic protist cells with a synchrotron X-ray fluorescence microprobe. Anal. Chem. 75 (15), 3806-3816 (2003).
- Chen, S., et al. The Bionanoprobe: hard X-ray fluorescence nanoprobe with cryogenic capabilities. J Synchrotron Radiat. 21, 66-75 (2014).
- Medalia, O., et al. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science. 298 (5596), 1209-1213 (2002).
- Forster, F., Medalia, O., Zauberman, N., Baumeister, W., Fass, D. Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography. Proc. Natl. Acad. Sci. U. S. A. 102 (13), 4729-4734 (2005).
- Muller, M., Moor, H. . Science of Biological Specimen. , 131-138 (1984).
- Moor, H., Riehle, U. . Proceedings of the 4th Eur. Reg. Conference Electron. , 33-34 (1968).
- Sitte, H. Advanced instrumentation and methodology related to cryoultramicrotomy: a review. Scanning Microsc Suppl. 10, 387-463 (1996).
- Studer, D., Graber, W., Al-Amoudi, A., Eggli, P. A new approach for cryofixation by high-pressure freezing. J. Microsc. 203, (Pt. 3, 285-294 (2001).
- Matsuyama, S., et al. Elemental mapping of frozen-hydrated cells with cryo-scanning x-ray fluorescence microscopy). X-Ray Spectrom. 39, 260-266 (2010).
- Schrag, M., et al. The effect of formalin fixation on the levels of brain transition metals in archived samples. Biometals. 23 (6), 1123-1127 (2010).
- James, S. A., et al. Quantitative comparison of preparation methodologies for X-ray fluorescence microscopy of brain tissue. Analytical and Bioanalytical Chemistry. 401 (3), 853-864 (2011).
- Al-Amoudi, A., et al. Cryo-electron microscopy of vitreous sections. EMBO J. 23 (18), 3583-3588 (2004).
- Bouchet-Marquis, C., Hoenger, A. Cryo-electron tomography on vitrified sections: a critical analysis of benefits and limitations for structural cell biology. Micron. 42 (2), 152-162 (2011).
- Bouchet-Marquis, C., Dubochet, J., Fakan, S. Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture. Histochem. Cell Biol. 125 (1-2), 1-2 (2006).
- Mesman, R. J. A novel method for high-pressure freezing of adherent cells for frozen hydrated sectioning and CEMOVIS. J. Struct. Biol. 183 (3), 527-530 (2013).
- Hackett, M. J., et al. Chemical Alterations to murine brain tissue induced by formalin fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis. Analyst. 136 (14), 2941-2952 (2011).
- Vogt, S. MAPS: A set of software tools for analysis and visualization of 3D x-ray fluorescence data sets. J Phys IV France. 104, 635-638 (2003).
- Vantelon, D., Lanzirotti, A., Scheinost, A. C., Kretzschmar, R. Spatial distribution and speciation of lead around corroding bullets in a shooting range soil studied by micro-X-ray fluorescence and absorption spectroscopy. Environ. Sci. Technol. 39 (13), 4808-4815 (2005).
- Robison, G., et al. X-ray fluorescence imaging of the hippocampal formation after manganese exposure. Metallomics : Integrated Biometal Science. 5 (11), 1554-1565 (2013).
- Hard Kemner, K. M. X-ray micro(spectro)scopy: a powerful tool for the geomicrobiologists. Geobiology. 6 (3), 270-277 (2008).
- Walsh, W. Scientific Testing of Beethoven’s Hair. 17, (2000).
- Casadio, F., Rose, V. High-resolution fluorescence mapping of impurities in historical zinc oxide pigments: hard X-ray nanoprobe applications to the paints of Pablo Picasso. Applied Physics A: Materials Science and Processing. 111 (1), 1-8 (2013).
- Leonardo, T., et al. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques–subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis. Metallomics : Integrated Biometal Science. 6 (2), 316-329 (2014).
- Wang, P., et al. Quantitative determination of metal and metalloid spatial distribution in hydrated and fresh roots of cowpea using synchrotron-based X-ray fluorescence microscopy. Sci. Total Environ. , 463-464 (2013).
- Ducic, T., et al. X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons. J. Neurochem. 124 (2), 250-261 (2013).
- Kim, A. M., Vogt, S., O’Halloran, T. V., Woodruff, T. K. Zinc availability regulates exit from meiosis in maturing mammalian oocytes. Nature Chemical Biology. 6 (9), 674-681 (2010).
- Ortega, R., Cloetens, P., Deves, G., Carmona, A., Bohic, S. Iron storage within dopamine neurovesicles revealed by chemical nano-imaging. PloS One. 2 (9), (2007).
- Bohic, S., et al. Synchrotron hard X-ray microprobe: fluorescence imaging of single cells. Appl. Phys. Lett. 78 (22), 3544-3546 (2001).
- Kosior, E., et al. Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification. J. Struct. Biol. 177 (2), 239-247 (2012).
- Glesne, D., Vogt, S., Maser, J., Legnini, D., Huberman, E. Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells. J. Struct. Biol. 155 (1), 2-11 (2006).
- McRae, R., Lai, B., Vogt, S., Fahrni, C. J. Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles. J. Struct. Biol. 155 (1), 22-29 (2006).
- Yang, L., et al. Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy. Proc. Natl. Acad. Sci. U. S. A. 102 (32), 11179-11184 (2005).
- Wagner, D., et al. Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal system. J. Immunol. 174 (3), 1491-1500 (2005).
- Harris, H. H., et al. Time-dependent uptake, distribution and biotransformation of chromium(VI) in individual and bulk human lung cells: application of synchrotron radiation techniques. Journal Of Biological Inorganic Chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 10 (2), 105-118 (2005).
- Corezzi, S., et al. Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots. Anal. Biochem. 388 (1), 33-39 (2009).
- Marmorato, P., et al. Cellular distribution and degradation of cobalt ferrite nanoparticles in Balb/3T3 mouse fibroblasts. Toxicol. Lett. 207 (2), 128-136 (2011).
- Weekley, C. M., et al. distribution, and speciation of selenoamino acids by human cancer cells: X-ray absorption and fluorescence methods. Biochimie. 50 (10), 1641-1650 (2011).
- Yuan, Y., et al. Epidermal growth factor receptor targeted nuclear delivery and high-resolution whole cell X-ray imaging of Fe3O4@TiO2 nanoparticles in cancer cells. ACS Nano. 7 (12), 10502-10517 (2013).
- McRae, R., Bagchi, P., Sumalekshmy, S., Fahrni, C. J. In situ imaging of metals in cells and tissues. Chem. Rev. 109 (10), 4780-4827 (2009).
- Carter, E. A., et al. Silicon nitride as a versatile growth substrate for microspectroscopic imaging and mapping of individual cells. Molecular Biosystems. 6 (7), 1316-1322 (2010).
