My group has developed a novel scanning transmission electron microscopy (STEM) technology to image whole eukaryotic cells in their native liquid state, so-called Liquid STEM, based on the technology of liquid-phase electron microscopy. Proteins are specifically labeled with electron dense nanoparticles. The atomic number (Z) contrast 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.
Three different setups are used. (1) The cells are fully enclosed in a microfluidic chamber with two SiN windows. Imaging is accomplished with dedicated STEM at 200 keV electron beam energy. (2) 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. (3) A sample, for example, a layer of cells in liquid is covered with a graphene sheet protecting the liqudi from evaporating, and the sample is then imaged with high resolution dedicated STEM at 200 keV. See: Dahmke et al. ACS Nano 11, 11108-11117, 2017. link
The Liquid STEM approach combines much of the functionality of light microscopy with the high spatial resolution of electron microscopy. Our latest research involves the combination of fluorescence microscopy with Liquid STEM using proteins labeled with quantum dots. We have extensively studied the physics of image formation of STEM in thick layers of liquid. Liquid STEM of fully hydrated live yeast cells was demonstrated feasible.
Liquid STEM is also 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.
See also: de Jonge & Ross, Nat. Nanotechnol. 6, 695-704, 2011. link
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