My team has developed a novel electron microscopy technology to image whole eukaryotic cells in their native liquid state, so-called liquid-phase electron microscopy (LP-EM). Proteins are specifically labeled with electron dense nanoparticles. The atomic number (Z) contrast of scanning transmission electron microscopy (STEM) is then used to image the nanoparticles within a layer of liquid containing the cells. Labels of different sizes and compositions can be distinguished. LP-EM is also used to study protein complexes and nanomaterials in liquid.
Three different setups are used:
- The cells are fully enclosed in a microfluidic chamber with two SiN windows. Imaging is accomplished with STEM at 200 keV electron beam energy. See: de Jonge et al. 2009
- The sample is maintained in a saturated water vapor atmosphere, while a thin layer of water covers the cell. Environmental scanning electron microscopy (ESEM) with STEM detection is used for imaging. See: Peckys et al. 2013
- A sample in liquid is covered with a graphene sheet protecting the liquid from evaporating, and the sample is then imaged with STEM at 200 keV. See: Dahmke et al. 2017
The LP-EM approach combines much of the functionality of light microscopy with the high spatial resolution of electron microscopy. Correlative fluorescence microscopy and LP-EM is possible using quantum dot nanoparticles as protein labels. We have extensively studied the physics of image formation of STEM in thick layers of liquid (see: de Jonge, 2018 and de Jonge et al. 2019). We demonstrated that graphene enclosure of a protein sample increased the electron dose tolerance by an order of magnitude compared to cryo electron microscopy (see: Keskin & de Jonge 2018)
LP-EM has also been used to study nanomaterials in liquid to explore, for example, nanoparticle movement in thin liquid layers, self-assembly processes, and growth of nanomaterials in liquid.
A review about LP-EM is available here: de Jonge & Ross, 2011
The primary method currently used for obtaining insight into the three-dimensional (3D) organization of cellular structures is tilt-series transmission electron microscopy (TEM). However, its application is limited on account of the high tilt-angles of up to 70°, and it is a challenge to image micrometers-thick samples containing, for example, whole cells. We have developed a novel 3D STEM technique for cell biology obtaining nanometer resolution on biological specimens. Aberration-corrected 3D STEM is capable of high-resolution 3D imaging without a tilt stage. In a manner similar to confocal light microscopy, the sample is scanned layer-by-layer by changing the objective lens focus so that a focal series is recorded. Nanoscale 3D resolution results from the high beam convergence angle. One of our future aims is to obtain 3D information from whole cells in liquid. We are currently improving the vertical resolution by combining focal- and tilt-series STEM. We have recently demonstrated that the combined tilt- and focal series leads to an improved 3D reconstruction with information in the missing wedge compared to tilt-series only. This research is conducted together with Dr. Tim Dahmen of the German Center for Artificial Intelligence and funded by the DFG in the project: “TFS-STEM: Combined tilt- and focal series for STEM tomography with a computational correction for beam blurring.”
See also: Dahmen et al., Microsc. Microanal. 20, 548-560, 2014. link