Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy

We describe the use of transmission electron microscopy (TEM) for cellular ultrastructural examination of nanoparticle (NP)-exposed biomaterials. Preparation and imaging of electron-transparent thin cell sections with TEM provides excellent spatial resolution ( ∼ 1 nm), which is required to track these elusive materials. This protocol provides a step-by-step method for the mass-basis dosing of cultured cells with NPs, and the process of fixing, dehydrating, staining, resin embedding, ultramicrotome sectioning and subsequently visualizing NP uptake and translocation to specific intracellular locations with TEM. In order to avoid potential artifacts, some technical challenges are addressed. Based on our results, this procedure can be used to elucidate the intracellular fate of NPs, facilitating the development of biosensors and therapeutics, and provide a critical component for understanding NP toxicity. This protocol takes ∼ 1 week.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

265,23 € per year

only 22,10 € per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

Nanoscale observation of PM2.5 incorporated into mammalian cells using scanning electron-assisted dielectric microscope

Article Open access 08 January 2021

In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells

Article 13 November 2023

Nanoparticle corona artefacts derived from specimen preparation of particle suspensions

Article Open access 24 March 2020

References

  1. Colvin, V.L. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol.21, 1166 (2003). ArticleCASPubMedGoogle Scholar
  2. Hoet, P.H., Nemmar, A. & Nemery, B. Health impact of nanomaterials? Nat. Biotechnol.22, 19 (2004). ArticleCASPubMedGoogle Scholar
  3. Maynard, A.D. et al. Safe handling of nanotechnology. Nature444, 267 (2006). ArticleCASPubMedGoogle Scholar
  4. Nel, A., Xia, T., Madler, L. & Li, N. Toxic potential of materials at the nanolevel. Science311, 622 (2006). ArticleCASPubMedGoogle Scholar
  5. Hussain, S.M., Hess, K.L., Gearhart, J.M., Geiss, K.T. & Schlager, J.J. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol. In Vitro19, 975–983 (2005). ArticleCASPubMedGoogle Scholar
  6. Braydich-Stolle, L., Hussain, S., Schlager, J.J. & Hofmann, M.-C. In vitro cytotoxicity of nanoparticles in mammalian germ-line stem cells. Tox. Sci.88, 412–419 (2005). ArticleCASGoogle Scholar
  7. Hussain, S. et al. The interaction of manganese nanotubes with PC-12 cells induces dopamine depletion. J. Tox. Sci.92, 456–463 (2006). ArticleCASGoogle Scholar
  8. Skebo, J.E., Grabinski, C.M., Schrand, A.M., Schlager, J.J. & Hussain, S.M. Assessment of metal nanoparticle agglomeration, uptake, and interaction using a high illuminating system. Int. J. Tox.26, 135–141 (2007). ArticleCASGoogle Scholar
  9. Schrand, A.M. et al. Are diamond nanoparticles cytotoxic? J. Phys. Chem. B.111, 2–7 (2007a). ArticleCASPubMedGoogle Scholar
  10. Schrand, A.M., Dai, L., Schlager, J.J., Hussain, S.M. & Osawa, E. Differential biocompatibility of carbon nanotubes and nanodiamonds. Diam. Relat. Mater.16, 2118–2123 (2007b). ArticleCASGoogle Scholar
  11. Schrand, A.M. et al. Interaction and biocompatibility of multi-walled carbon nanotubes in PC-12 cells. Int. J. Neuroprot. Neuroregener.3, 115–121 (2007c). CASGoogle Scholar
  12. Wagner, A.J. et al. Cellular interaction of different forms of aluminum nanoparticles in rat alveolar macrophages. J. Phys. Chem. B.111, 7353–7359 (2007). ArticleCASPubMedGoogle Scholar
  13. Schrand, A.M., Braydich-Stolle, L.K., Schlager, J.J., Dai, L. & Hussain, S.M. Can silver nanoparticles be useful as potential biological labels? Nanotechnology19, 1–13 (2008a). ArticleCASGoogle Scholar
  14. Carlson, C. et al. Uniques cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. J. Phys. Chem. B.112, 13608–13619 (2008). ArticleCASPubMedGoogle Scholar
  15. Murdock, R.C., Braydich-Stolle, L., Schrand, A.M., Schlager, J.J. & Hussain, S.M. Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Tox. Sci.101, 239–253 (2008). ArticleCASGoogle Scholar
  16. Braydich-Stolle, L.K. et al. Crystal structure mediates mode of cell death in TiO2 nanotoxicity. J. Nanopart. Res.11, 1361–1374 (2008). ArticleCASGoogle Scholar
  17. Yu, K.O. et al. Toxicity of amorphous silica nanoparticles in mouse keratinocytes. J. Nanopart. Res.11, 15–24 (2009). ArticleCASGoogle Scholar
  18. Hussain, S.M. et al. Toxicity evaluation for safe use of nanomaterials: recent achievements and technical challenges. Adv. Mat.21, 1–11 (2009). ArticleCASGoogle Scholar
  19. Schrand, A.M., Citan, S.A. & Shenderova, O.A. Nanodiamond particles: properties and perspectives for bioapplications. Crit. Rev. Solid State Mater. Sci.34, 18–74 (2009). ArticleCASGoogle Scholar
  20. Colvin, V. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol.21, 1166–1170 (2003). ArticleCASPubMedGoogle Scholar
  21. Nel, A.E. et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater.8, 543–557 (2009). ArticleCASPubMedGoogle Scholar
  22. Ryman-Rasmussen, J.P., Riviere, J.E. & Monteiro-Rivere, N.A. Surface coatings determine cytotoxicity and irritation potential of quantum dot nanoparticles in epidermal keratinocytes. Soc. Invest. Derm.127, 143–153 (2006). ArticleCASGoogle Scholar
  23. Zhang, L.W., Zeng, L., Barron, A.R. & Monteiro-Riviere, N.A. Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes. Tox. Appl. Pharm.228, 200–211 (2008). ArticleCASGoogle Scholar
  24. Zhang, L.W. & Monteiro-Riviere, N.A. Mechanisms of quantum dot nanoparticle cellular uptake. Tox. Sci.110, 138–155 (2009). ArticleCASGoogle Scholar
  25. de Jonge, N., Peckys, D.B., Kremers, G.J. & Pistona, D.W. Electron microscopy of whole cells in liquid with nanometer resolution. Proc. Natl. Acad. Sci. USA106, 2159–2164 (2009). ArticlePubMedPubMed CentralGoogle Scholar
  26. Mayhew, T.M., Mühlfeld, C., Vanhecke, D. & Ochs, M. A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs. Ann. Anat.191, 153–170 (2009). ArticleCASPubMedGoogle Scholar
  27. Allen, T.D. et al. Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells. Nat. Protoc.2, 1180–1184 (2007). ArticleCASPubMedGoogle Scholar
  28. Allen, T.D. et al. A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Nat. Protoc.2, 1166–1172 (2007). ArticleCASPubMedGoogle Scholar
  29. Allen, T.D. et al. Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro–formed nuclei for western blotting and scanning electron microscopy (SEM). Nat. Protoc.2, 1173–1179 (2007). ArticleCASPubMedGoogle Scholar
  30. Wilhelm, C., Gazeau, F., Roger, J., Pons, J.N. & Bacri, J.C. Interaction of anionic superparamagnetic nanoparticles with cells: kinetic analyses of membrane adsorption and subsequent internalization. Langmuir18, 8148–8155 (2002). ArticleCASGoogle Scholar
  31. Conner, S.D. & Schmid, S.L. Regulated portals of entry into the cell. Nature422, 37–44 (2003). ArticleCASPubMedGoogle Scholar
  32. Shukla, R. et al. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview. Langmuir21, 10644–10654 (2005). ArticleCASPubMedGoogle Scholar
  33. Kam, N.W.S., Liu, Z. & Dai, H. Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway. Angew. Chem. Int. Ed.45, 577–581 (2006). ArticleCASGoogle Scholar
  34. Dobrovolskaia, M.A. & McNeil, S.E. Immunological properties of engineered nanomaterials. Nat. Nanotech.2, 469–478 (2007). ArticleCASGoogle Scholar
  35. Verma, A. et al. Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles. Nat. Mater.7, 588–595 (2008). ArticleCASPubMedPubMed CentralGoogle Scholar
  36. Jin, H., Heller, D.A. & Strano, M.S. Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in HIH3T3 cells. Nano Lett.8, 1577–1585 (2008). ArticlePubMedGoogle Scholar
  37. Yu, J. et al. Effect of surface functionality of magnetic silica nanoparticles on the cellular uptake by Glioma cells in vitro. J. Mater. Chem.19, 1265–1270 (2009). ArticleCASGoogle Scholar
  38. Geiser, M. et al. Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ. Health Perspect.113, 1555–1560 (2005). ArticlePubMedPubMed CentralGoogle Scholar
  39. Porter, A.E. et al. Visualizing the uptake of C60 to the cytoplasm and nucleus of human monocyte-derived macrophage cells using energy-filtered transmission electron microscopy and electron tomography. Enivron. Sci. Technol.41, 3012–3017 (2007). ArticleCASGoogle Scholar
  40. Porter, A.E. et al. Direct imaging of single-walled carbon nanotubes in cells. Nat. Nanotechnol.2, 713–717 (2007). ArticleCASPubMedGoogle Scholar
  41. Cheng, C. et al. Toxicity and imaging of multi-walled carbon nanotubes in human macrophage cells. Biomaterials30, 4152–4160 (2009). ArticleCASPubMedGoogle Scholar
  42. Steinbrecht, R.A. Freeze-substitution and freeze-drying. in Cryotechniques in Biological Electron Microscopy (eds. Steinbrecht, R.A. & Zierold, K.) 149–172 (K. Springer-Verlag, Berlin, 1987).
  43. Parthasarathy, M.V. Chapter 5 freeze-substitution. Methods Cell Biol.49, 57–69 (1995). ArticleCASPubMedGoogle Scholar
  44. Lucic, V. et al. Multiscale imaging of neurons grown in culture: from light microscopy to cryoelectron tomography. J. Struct. Biol.160, 146–156 (2007). ArticlePubMedGoogle Scholar
  45. Satori, A. et al. Correlative microscopy: bridging the gap between fluorescence light microscopy and cryoelectron tomography. J. Struct. Biol.160, 135–145 (2007). ArticleGoogle Scholar
  46. Hess, M.W., Muller, M., Debbage, P.L., Vetterlein, M. & Pavelka, M. Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Gel apparatus. J. Struct. Biol.130, 63–72 (2000). ArticleCASPubMedGoogle Scholar
  47. Marko, M. Focused ion beam thinning of frozen hydrated biological specimens for cryoelectron microscopy. Nat. Methods4, 215–217 (2007). ArticleCASPubMedGoogle Scholar
  48. Riley, M.R. et al. Comparison of the sensitivity of three lung derived cell lines to metals from combustion derived particulate matter. Toxicol. In Vitro19, 411–419 (2005). ArticleCASPubMedGoogle Scholar
  49. Ricarda-Lorenz, M. et al. Uptake of functionalized, fluorescent-labeled polymeric particles in different cell lines and stem cells. Biomaterials27, 2820–2828 (2006). ArticleCASGoogle Scholar
  50. Sayes, C.M., Reed, K.L. & Warheit, D.B. Assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles. Toxicol. Sci.97, 163–180 (2007). ArticleCASPubMedGoogle Scholar
  51. Chang, J.-S., Chang, K.L.B., Hwang, D.-F. & Kong, K.-L. In vitro cytotoxicity of silica nanoparticles at high concentrations strongly depends on the metabolic activity type of the cell line. Environ. Sci. Technol.41, 2064–2068 (2007). ArticleCASPubMedGoogle Scholar
  52. Xia, T. et al. Cationic polystyreen nanosphere toxicity depends on cell-specific endocytoic and mitohcondrial injury pathways. ACS Nano2, 85–96 (2008). ArticleCASPubMedGoogle Scholar
  53. Lanone, S. et al. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part. Fiber Toxicol.6, 14–26 (2009). ArticleCASGoogle Scholar
  54. Nabiev, I. et al. Non-functionalized nanocrystals can exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Nano Lett.7, 3452–3461 (2007). ArticleCASPubMedGoogle Scholar
  55. Krüger, A. et al. Unusually tight aggregation in detonation nanodiamond: identification and disintegration. Carbon43, 1722–1730 (2005). ArticleCASGoogle Scholar
  56. Greiner, N., Phillips, D., Johnson, J. & Volk, F. Dimaonds in detonation soot. Nature333, 440–442 (1998). ArticleGoogle Scholar
  57. Liang, Y., Ozawa, M. & Krueger, A. A general procedure to functionalize agglomerating nanoparticles demonstrated on nanodiamond. ACS Nano3, 2288–2296 (2009). ArticleCASPubMedGoogle Scholar
  58. Mei, B.C., Susumu, K., Medintz, I.L. & Mattoussi, H. Polyethylene glycol-based bidentate ligands to enhance quantum dot and gold nanoparticle stability in biological media. Nat. Protoc.4, 412–423 (2009). ArticleCASPubMedGoogle Scholar
  59. Monteiro-Riviere, N.A., Inman, A.O., Wang, Y.Y. & Nemanich, R.J. Surfactant effects on carbon nanotube interactions with human epidermal keratinocytes. Nanomedicine1, 293–299 (2005). ArticleCASPubMedGoogle Scholar
  60. Limbach, L.K. et al. Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Environ. Sci. Technol.39, 9370–9376 (2005). ArticleCASPubMedGoogle Scholar
  61. Shenderova, O. et al. Modification of detonation nanodiamonds by heat treatment in air. Diamond Relat. Mater.15, 1799 (2006). ArticleCASGoogle Scholar
  62. Morita, Y. et al. A facile and scalable process for size-controllable separation of nanodiamond particles as small as 4 nm. Small4, 2154–2157 (2008). ArticleCASPubMedGoogle Scholar
  63. Jamison, J.A. et al. Size dependent sedimentation properties of nanocrystals. ACS Nano2, 311–319 (2008). ArticleCASPubMedGoogle Scholar
  64. Teeguarden, J.G., Hinderliter, P.M., Orr, G., Thrall, B.D. & Pounds, J.G. Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments. Toxicol. Sci.95, 300–312 (2007). ArticleCASPubMedGoogle Scholar
  65. Jaiswal, J.K., Mattoussi, H., Mauro, J.M. & Simon, S.M. Long-term multiple color imaing of live cells using quantum dot bioconjugates. Nat. Biotechnol.21, 47–51 (2003). ArticleCASPubMedGoogle Scholar
  66. Monteiro-Riviere, N.A., Nemanich, R.J., Inman, A.O., Wang, Y.Y. & Riviere, J.E. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicol. Lett.155, 377–384 (2005). ArticleCASPubMedGoogle Scholar
  67. Soto, K., Garza, K.M. & Murr, L.E. Cytotoxic effects of aggregated nanomaterials. Acta Biomater.3, 351–358 (2007). ArticleCASPubMedGoogle Scholar
  68. Warheit, D.B., Webb, T.R., Sayes, C.M., Colvin, V.L. & Reed, K.L. Pulmonary instillation studies with nanoscale TiO2 rods and dots in rats: toxicity is not dependent upon particle size and surface area. Toxicol. Sci.91, 227–236 (2006). ArticleCASPubMedGoogle Scholar
  69. Stoeger, T. et al. Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice. Environ. Health Perspect.114, 328–333 (2006). ArticlePubMedGoogle Scholar
  70. Elder, A. et al. Effects of subchronically inhaled carbon black in three species. I. Retention kinetics, lung inflammation, and histopathology. Toxicol. Sci.88, 614–629 (2005). ArticleCASPubMedGoogle Scholar
  71. Brown, D.M., Wilson, M.R., MacNee, W., Stone, V. & Donaldson, K. Size dependent proinflammatory effects of ultrafine polystyrene particles: a role for surface area and oxidative stress in the enhanced activity of ultrafines. Toxicol. Appl. Pharmacol.175, 191–199 (2001). ArticleCASPubMedGoogle Scholar
  72. Chithrani, B.D. & Chan, W.C.W. Elucidating the mechanisms of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett.7, 1542–1550 (2007). ArticleCASPubMedGoogle Scholar
  73. Nativo, P., Prior, I.A. & Brust, M. Uptake and intracellular fate of surface-modified gold nanoparticles. ACS Nano2, 1639–1644 (2008). ArticleCASPubMedGoogle Scholar
  74. Gopee, N.V. et al. Migration of intradermally injected quantum dots to sentinel organs in mice. Toxicol. Sci.98, 249–257 (2007). ArticleCASPubMedGoogle Scholar
  75. Gojova, A. et al. Induction of inflammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition. Environ. Health Perspect.115, 403–409 (2007). ArticleCASPubMedGoogle Scholar
  76. Gratton, S.E. et al. The effect of particle design on cellular internalization pathways. Proc. Natl. Acad. Sci. USA105, 11613–11618 (2008). ArticlePubMedPubMed CentralGoogle Scholar
  77. Vincent, A. et al. Protonated nanoparticle surface governing ligand tethering and cellular targeting. ACS Nano3, 1203–1211 (2009). ArticleCASPubMedPubMed CentralGoogle Scholar
  78. Karnovsky, M.J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. JCB27, 137A–138A (1965). Google Scholar
  79. McDowell, E.M. & Trump, B.F. Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med.100, 405–414 (1976). CASPubMedGoogle Scholar
  80. Monteiro-Riviere, N. & Inman, A. Challenges for assessing carbon nanomaterial toxicity to the skin. Carbon44, 1070–1078 (2006). ArticleCASGoogle Scholar
  81. Rouse, J.G., Yang, J., Barron, A.R. & Monteiro-Riviere, N.A. Fullerene-based amino acid nanoparticle interactions with human epidermal keratinocytes. Toxicol. In Vitro20, 1313–1320 (2006). ArticleCASPubMedGoogle Scholar
  82. Zhang, L.W., Zeng, L., Barron, A.R. & Monteiro-Riviere, N.A. Biological interactions of functionalized single-wall carbon nanotubes in human epidermal keratinocytes. Int. J. Toxicol.26, 103–113 (2007). ArticleCASPubMedGoogle Scholar
  83. Zhang, Y. & Zhang, J. Surface modification of monodisperse magnetite nanoparticles for improved intracellular uptake to breast cancer cells. J. Colloid Interface Sci.283, 352–357 (2005). ArticleCASPubMedGoogle Scholar
  84. Millonig, G. Advantages of a phosphate buffer for osmium tetroxide solutions in fixation. J. Appl. Phys.32, 1637 (1961). Google Scholar
  85. Reynolds, E.S. Use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol.17, 208–212 (1963). ArticleCASPubMedPubMed CentralGoogle Scholar
  86. Graham, L. & Orenstein, J.M. Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research. Nat. Protoc.2, 2439–2450 (2007). ArticleCASPubMedPubMed CentralGoogle Scholar
  87. Robards, A.W. & Wilson, A.J. Procedure in Electron Microscopy: Module 5:5 Basic Biological Preparation Techniques for TEM 5:5.1–5:5.28 (John Wiley & Son, Hoboken, NJ, 1999).
  88. Aderem, A. & Underhill, D.M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol.17, 593–623 (1999). ArticleCASPubMedGoogle Scholar

Acknowledgements

A.M.S. received funding from the National Research Council (NRC) Fellowship program funded by the Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD), a program administered by the Defense Threat Reduction Agency (DTRA).