Applied Microbiology and Biotechnology

, Volume 101, Issue 13, pp 5469–5479 | Cite as

Transmission electron microscopy artifacts in characterization of the nanomaterial-cell interactions

  • Yu Hang Leung
  • Mu Yao Guo
  • Angel P. Y. Ma
  • Alan M. C. Ng
  • Aleksandra B. Djurišić
  • Natalie Degger
  • Frederick C. C. Leung
Methods and protocols

Abstract

We investigated transmission electron microscopy artifacts obtained using standard sample preparation protocols applied to the investigation of Escherichia coli cells exposed to common nanomaterials, such as TiO2, Ag, ZnO, and MgO. While the common protocols for some nanomaterials result only in known issues of nanomaterial-independent generation of anomalous deposits due to fixation and staining, for others, there are reactions between the nanomaterial and chemicals used for post-fixation or staining. Only in the case of TiO2 do we observe only the known issues of nanomaterial-independent generation of anomalous deposits due to exceptional chemical stability of this material. For the other three nanomaterials, different artifacts are observed. For each of those, we identify causes of the observed problems and suggest alternative sample preparation protocols to avoid artifacts arising from the sample preparation, which is essential for correct interpretation of the obtained images and drawing correct conclusions on cell-nanomaterial interactions. Finally, we propose modified sample preparation and characterization protocols for comprehensive and conclusive investigations of nanomaterial-cell interactions using electron microscopy and for obtaining clear and unambiguous revelation whether the nanomaterials studied penetrate the cells or accumulate at the cell membranes. In only the case of MgO and ZnO, the unambiguous presence of Zn and Mg could be observed inside the cells.

Keywords

TEM SEM Nanoparticles Escherichia coli 

Notes

Acknowledgements

Financial support from the Strategic Research Theme, University Development Fund, and Seed Funding of the University of Hong Kong are acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This paper does not contain any studies with human participants. Ethics approval by The Committee for the Use of Live Animals in Teaching and Research (CULATR 2781-12), the University of Hong Kong, with a valid Cap.340 license was obtained for animal experiments on fish embryos.

Supplementary material

253_2017_8305_MOESM1_ESM.pdf (305 kb)
ESM 1 (PDF 304 kb)

References

  1. Albanese A, Chan WC (2011) Effect of gold nanoparticle aggregation on cell uptake and toxicity. ACS Nano 5:5478–5489. doi: 10.1021/nn2007496 CrossRefPubMedGoogle Scholar
  2. Applerot G, Lipovsky A, Dror R, Perkas N, Nitzan Y, Lubart R, Gedanken A (2009) Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS-mediated cell injury. Adv Funct Mater 19:842–852. doi: 10.1002/adfm.200801081 CrossRefGoogle Scholar
  3. Asharani PV, Wu YL, Gong Z, Valiyaveettil S (2008) Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19:255102. doi: 10.1088/0957-4484/19/25/255102 CrossRefPubMedGoogle Scholar
  4. Brayner R, Ferrari-Iliou R, Brivois N, Djediat S, Benedetti MF, Fievet F (2006) Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nano Lett 6:866–870. doi: 10.1021/nl052326h CrossRefPubMedGoogle Scholar
  5. Djurišić AB, Leung YH, Ng AMC, Xu XY, Lee PKH, Degger N, Wu RSS (2015) Toxicity of metal oxide nanoparticles: mechanisms, characterization, and avoiding experimental artefacts. Small 11:26–44. doi: 10.1002/smll.201303947 CrossRefPubMedGoogle Scholar
  6. Egerton RF (2014) Choice of operating voltage for a transmission electron microscope. Ultramicroscopy 145:85–93. doi: 10.1016/j.ultramic.2013.10.019 CrossRefPubMedGoogle Scholar
  7. Fernandez-Segura E, Warley A (2008) Electron probe X-ray microanalysis for the study of cell physiology. Methods Cell Biol 88:19–43. doi: 10.1016/S0091-679X(08)00402-0 CrossRefPubMedGoogle Scholar
  8. Gillespie RB, Baumann PC (1986) Effects of high tissue concentrations of selenium on reproduction by bluegills. Trans Am Fish Soc 115:208–213. doi: 10.1577/1548-8659(1986)1153C208:EOHTCO3E2.0.CO;2 CrossRefGoogle Scholar
  9. He XJ, Aker WG, Hwang HM (2014) An in vivo study on the photo-enhanced toxicities of S-doped TiO2 nanoparticles to zebrafish embryos (Danio rerio) in terms of malformation, mortality, rheotaxis dysfunction, and DNA damage. Nanotoxicology 8:185–195. doi: 10.3109/17435390.2013.874050 CrossRefPubMedGoogle Scholar
  10. Hernández-Moreno D, Li LXY, Connolly M, Conde E, Fernández M, Schuster M, Navas JM, Fernández-Cruz M (2016) Mechanisms underlying the enhancement of toxicity caused by the coincubation of zinc oxide and copper nanoparticles in a fish hepatoma cell line. Environ Toxicol Chem 35:2562–2570. doi: 10.1002/etc.3425 CrossRefPubMedGoogle Scholar
  11. Huang Z, Zheng X, Yan D, Yin G, Liao X, Kang Y, Yao Y, Huang D, Hao B (2008) Toxicological effect of ZnO nanoparticles based on bacteria. Langmuir 24:4140–4144. doi: 10.1021/la7035949 CrossRefPubMedGoogle Scholar
  12. Ji Z, Wang X, Zhang H, Lin S, Meng H, Sun B, George S, Xia T, Nel AE, Zink JI (2012) Designed synthesis of CeO2 nanorods and nanowires for studying toxicological effects of high aspect ratio nanomaterials. ACS Nano 6:5366–5380. doi: 10.1021/nn3012114 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Jiang W, Mashayekhi H, Xing B (2009) Bacterial toxicity comparison between nano- and micro-scaled oxide particles. Environ Pollut 157:1619–1625. doi: 10.1016/j.envpol.2008.12.025 CrossRefPubMedGoogle Scholar
  14. Johnston TA, Bodaly RA, Latif MA, Fudge RJP, Strange NE (2001) Intra- and interpopulation variability in maternal transfer of mercury to eggs of walleye (Stizostedion vitreum). Aquat Toxicol 52:73–85. doi: 10.1016/S0166-445X(00)00129-6 CrossRefPubMedGoogle Scholar
  15. Kumar A, Pandey AK, Singh SS, Shanker R, Dhawan A (2011) Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells. Chemosphere 83:1124–1132. doi: 10.1016/j.chemosphere.2011.01.025 CrossRefPubMedGoogle Scholar
  16. Leung YH, Ng AMC, Xu XY, Shen ZY, Gethings LA, Wong MT, Chan CMN, Guo MY, Ng YH, Djurišić AB, Lee PKH, Chan WK, Yu LH, Phillips DL, Ma APY, Leung FCC (2014) Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of MgO nanoparticles towards Escherichia coli. Small 10:1171–1183. doi: 10.1002/smll.201302434 CrossRefPubMedGoogle Scholar
  17. Li WR, Xie XB, Shi QS, Zeng HY, You-Sheng OY, Chen YB (2010) Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Appl Microbiol Biotechnol 85:1115–1122. doi: 10.1007/s00253-009-2159-5 CrossRefPubMedGoogle Scholar
  18. Maurer-Jones MA, Gunsolus IL, Meyer BM, Christenson CJ, Haynes CL (2013) Impact of TiO2 nanoparticles on growth, biofilm formation, and flavin secretion in Shewanella oneidensis. Anal Chem 85:5810–5818. doi: 10.1021/ac400486u CrossRefPubMedPubMedCentralGoogle Scholar
  19. Müller KH, Kulkarni J, Motskin M, Goode A, Winship P, Skepper JN, Ryan MP, Porter AE (2010) pH-dependent toxicity of high aspect ratio ZnO nanowires in macrophages due to intracellular dissolution. ACS Nano 4:6767–6779. doi: 10.1021/nn101192z CrossRefPubMedGoogle Scholar
  20. Pan X, Wang Y, Chen Z, Pan D, Cheng Y, Liu Z, Lin Z, Guan X (2013) Investigation of antibacterial activity and related mechanism of a series of nano-Mg(OH)2. ACS Appl Mater Interfaces 5:1137–1142. doi: 10.1021/am302910q CrossRefPubMedGoogle Scholar
  21. Pelletier DA, Suresh AK, Holton GA, McKeown CK, Wang W, Gu B, Mortensen NP, Allison DP, Joy DC, Allison MR, Brown SD, Phelps TJ, Doktycz MJ (2010) Effects of engineered cerium oxide nanoparticles on bacterial growth and viability. Appl Environ Microbiol 76:7981–7989. doi: 10.1128/AEM.00650-10 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Penen F, Malherbe J, Isaure MP, Dobritzsch D, Bertalan I, Gontier E, Coustumer PL, Schaumlöffel D (2016) Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS. J Trace Elem Med Biol 37:62–68. doi: 10.1016/j.jtemb.2016.04.014 CrossRefPubMedGoogle Scholar
  23. Schrand AM, Schlager JJ, Dai L, Hussain SM (2010) Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy. Nat Protoc 5:744–757. doi: 10.1038/nprot.2010.2 CrossRefPubMedGoogle Scholar
  24. Scown TM, Santos EM, Johnston BD, Gaiser B, Baalousha M, Mitov S, Lead JR, Stone V, Fernandes TF, Jepson M, van Aerle R, Tyler CR (2010) Effects of aqueous exposure to silver nanoparticles of different sizes in rainbow trout. Toxicol Sci 115:521–534. doi: 10.1093/toxsci/kfq076 CrossRefPubMedGoogle Scholar
  25. Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ (2002) Metal oxide nanoparticles as bactericidal agents. Langmuir 18:6679–6686. doi: 10.1021/la0202374 CrossRefGoogle Scholar
  26. Thill A, Zeyons O, Spalla O, Chauvat F, Rose J, Auffan M, Flank AM (2006) Cytotoxicity of CeO2 nanoparticles for Escherichia coli. Physico-chemical insight of the cytotoxicity mechanism. Environ Sci Technol 40:6151–6156. doi: 10.1021/es060999b CrossRefPubMedGoogle Scholar
  27. Tu Y, Lv M, Xiu P, Huynh T, Zhang M, Castelli M, Liu Z, Huang Q, Fan C, Fang H, Zhou R (2013) Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets. Nat Nanotechnol 8:594–601. doi: 10.1038/nnano.2013.125 CrossRefPubMedGoogle Scholar
  28. Yang Y, Zhu H, Colvin VL, Alvarez PJ (2011) Cellular and transcriptional response of Pseudomonas stutzeri to quantum dots under aerobic and denitrifying conditions. Environ Sci Technol 45:4988–4994. doi: 10.1021/es1042673 CrossRefPubMedGoogle Scholar
  29. Yeo MK, Park HG (2012) Gene expression in zebrafish embryos following exposure to Cu-doped TiO2 and pure TiO2 nanometer-sized photocatalysts. Mol Cell Toxicol 8:127–137. doi: 10.1007/s13273-012-0016-6 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Yu Hang Leung
    • 1
  • Mu Yao Guo
    • 1
    • 2
  • Angel P. Y. Ma
    • 3
  • Alan M. C. Ng
    • 1
    • 2
  • Aleksandra B. Djurišić
    • 1
  • Natalie Degger
    • 3
  • Frederick C. C. Leung
    • 3
  1. 1.Department of PhysicsUniversity of Hong KongPokfulam RoadHong Kong
  2. 2.Department of PhysicsSouthern University of Science and Technology of ChinaShenzhenChina
  3. 3.School of Biological SciencesUniversity of Hong KongPokfulam RoadHong Kong

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