| AVS 58th Annual International Symposium and Exhibition | |
| In Situ Spectroscopy and Microscopy Focus Topic | Tuesday Sessions |
| Session IS+AS+SS-TuM |
| Session: | In Situ Studies of Organic and Soft Materials and Liquid-Solid Interfaces |
| Presenter: | Diana Peckys, Vanderbilt University School of Medicine |
| Authors: | D.B. Peckys, Vanderbilt University School of Medicine N. De Jonge, Vanderbilt University School of Medicine |
| Correspondent: | Click to Email |
We have applied a novel electron microscopy technique, referred to as liquid scanning transmission electron microscopy (liquid STEM) [1, 2] for the imaging of live eukaryotic cells. In two separate experiments, we studied a) nano particle (NP) uptake in COS-7 cells [3], a green monkey kidney fibroblast cell line, and, b) the ultrastructure of Schizosaccharomyces pombe cells [4], also known as fission yeast. The cells were confirmed to be alive at the onset of the liquid STEM imaging using specific fluorescent, live indicating dyes and correlative fluorescence microscopy. For the STEM imaging in liquid the cells were placed (in liquid) in a microfluidic chamber. The chamber had two ultra-thin electron-transparent windows allowing the passage of electrons and photons. The dimensions of the COS-7 cells required a thicker liquid filled space in the microfluidic chamber compared to the experiments with the fission yeast cells, and contrast was mainly obtained on the gold NP’s. However, the thinner S. pombe cells allowed a thinner liquid layer, and images were recorded of the cellular ultrastructure.
Despite the fact, that the cells were not anymore alive after the STEM imaging, we consider the first STEM images taken from a cell or a specific cellular region, to represent the unperturbed and therefore physiological state. We derived this assumption after evaluation of the STEM images for signs of radiation damage at the achieved resolution. Our STEM results were found to be consistent with known data about intracellular NP trafficking and storage in mammalian cells and data about the dimensions and distribution of organelles in fission yeast.
In conclusion, we have demonstrated the feasibility of STEM imaging live eukaryotic cells. The advantages of this approach are a) a several-fold higher resolution than live cell imaging with conventional light microscopy, b) a much faster (hours versus days) sample preparation than needed for conventional transmission electron microscopy (TEM) imaging of cells, c) absence of artifact introduction associated with conventional TEM sample preparation, and d) no need for introducing any kind of labels in order to achieve a similar range of resolution as possible with the new nanoscopic imaging techniques.
References
[1] de Jonge, N., Peckys, D.B., Kremers, G.J. & Piston, D.W., Proc. Natl. Acad. Sci. 106, 2159-2164, 2009.
[2] Peckys, D.B., Veith, G.M., Joy, D.C. & de Jonge, N., PLoS One 4, e8214-1-7, 2009.
[3] Peckys, D.B. & N. de Jonge, Nano Lett. 11, 1733-1738, 2011.
[4] Peckys, D.B., Mazur, P., Gould, K.L. & de Jonge, N., Biophys. J., in press, 2011.