It is now possible to study biological systems (e.g. eukaryotic cells) at nanometer spatial resolution in their native liquid environment using electron microscopy [1-3]. In contrast to conventional procedures, these new methods do not involve complex preparation steps like embedding, cutting, or freeze-sectioning. It is further possible to minimize negative drying artifacts by adjusting experimental conditions that represent the native state as close as possible, i.e. the presence of water. Several different approaches meeting these requirements have been developed to study whole cells of different sizes:

Method 1: Two silicon microchips with electron transparent windows can be used to realize a microfluidic chamber that is sealed from the vacuum in the electron microscope (Figure 1). The chamber between the two microchips is thick enough to contain whole cells, yet thin enough to ensure a sufficient transmission of electrons. It is further possible to create bubbles in the liquid cell compartment by inducing a high density of electron dose on the sample. This increases the obtained contrast due to the minimized amount of scattering solvent. The liquid cell compartment can be used in a standard Transmission electron microscope (TEM). Method 2: Environmental scanning electron microscopy (ESEM) can be used to study cells covered in a thin layer of liquid surrounded by a saturated water atmosphere. The thickness of the liquid layer is controlled by adjusting the temperature and the environmental pressure within the vacuum chamber. Method 3: The wet sample is attached to an electron transparent support (e.g. graphene, carbon, silicon nitride) and covered with a thin membrane (e.g. mono or multilayer graphene) to realize a thin layer of liquid around the sample [4]. This method allows for high resolution imaging as the amount of solvent is reduced to a minimum. It can be used in any conventional TEM and SEM with scanning transmission electron microscopy (STEM) detection. The presence of water can be confirmed by beam-induced bubbles.

Each of these three methods can be used to study the location and stoichiometry of transmembrane proteins within the intact plasma membrane [2, 3] with relevance to cancer research [5]. Nanoparticles, specifically attached to proteins, provide enough contrast for imaging (Figure 2).  Also correlative light- and electron microscopy is readily possible, so that large numbers of cells can be screened, while selected regions can be studied with high resolution [5].

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., Baudoin, J.P., Eder, M., Werner, U., de Jonge, N., Scientific reports 3, 2626, 2013.

[3]       Peckys, D.B., de Jonge, N., Microscopy and microanalysis 20, 346-365, 2014.

[4]       Walker, M. I.; Weatherup, R. S.; Bell, N. A. W.; Hofmann, S.; Keyser, U. F. Appl. Phys. Lett. 2015, 106, 023119.

[5]       Peckys, D.B., Korf, U., de Jonge, N., Sci Adv 1, e1500165, 2015.

Acknowledgements: We thank E. Arzt for his support through INM.  Research in part supported by the Leibniz Competition 2014. R.S.W. acknowledges a Research Fellowship from St. John’s College, Cambridge and a Marie Skłodowska-Curie Individual Fellowship (Global) under grant ARTIST (no. 656870) from the European Union’s Horizon 2020 research and innovation programme.