Niels de Jonge

Three-dimensional (3D) datasets were recorded of gold nanoparticles placed on both sides of silicon nitride membranes using focal series aberration-corrected scanning transmission electron microscopy (STEM). Deconvolution of the 3D datasets was applied to obtain the highest possible axial resolution. The deconvolution involved two different point spread functions, each calculated iteratively via blind deconvolution. Supporting membranes of different thicknesses were tested to study the effect of beam broadening on the deconvolution. It was found that several iterations of deconvolution was efficient in reducing the imaging noise. With an increasing number of iterations, the axial resolution was increased, and most of the structural information was preserved. Additional iterations improved the axial resolution by maximal a factor of 4 to 6, depending on the particular dataset, and up to 8 nm maximal, but also led to a reduction of the lateral size of the nanoparticles in the image. Thus, the deconvolution procedure optimized for the highest axial resolution is best suited for applications where one is interested in the 3D locations of nanoparticles only.

Understanding the structure and dynamics of the protein complexes that underlie cellular function is a central scientific challenge. Biochemical techniques used to identify such complexes would be enhanced by the imaging of specific molecular positions in the context of intact cells, with protein-scale resolution (on the order of a few nanometers). Currently, though, nanometer resolution can only be achieved at the cost of less-direct imaging of the unperturbed cell. Cellular ultrastructure is traditionally studied by transmission electron microscopy (TEM), which yields nanometer resolution on embedded and stained sections, or cryo sections. These cellular samples are neither intact nor in their native liquid state. Light microscopy is used to image protein distributions in fluorescently labeled cells in liquid to investigate cellular function, but even recent improvements in resolution by nanoscopy techniques are still insufficient to resolve the individual constituents of protein complexes. Thus, development of techniques capable of high-resolution imaging in native cellular states would contribute significantly to our understanding of cellular function at the molecular level. The development of liquid compartments that include electron-transparent silicon nitride membrane windows has led to the introduction of a novel concept to achieve nanometer resolution on tagged proteins in cells.

Silicon microchips with thin, electron transparent silicon nitride windows provide a sample support that accommodates both light-, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and there is no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare the microchips for biological samples, and examples of images obtained using different light and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light and electron microscopy.

Solid materials in subambient gaseous environments have been imaged using in situ transmission electron microscopy (TEM), for example to study dynamic effects: carbon nanotube growth, nanoparticle changes during redox reactions, and phase transitions in nanoscale systems. In these studies the vacuum level in the specimen region of the electron microscope was increased to pressures of up to 10 mbar using pump-limiting apertures that separated the specimen region from the rest of the high-vacuum electron column, but it has not been possible to achieve the higher pressures that are desirable for catalysis research. TEM imaging at atmospheric pressure and at elevated temperature was achieved with 0.2-nm resolution by enclosing a gaseous environment several micrometers thick between ultra-thin, electron transparent silicon nitride windows. Although Ångström-level resolution in situ TEM has been demonstrated with aberration-corrected systems, the key difficulty with TEM imaging is its dependence on phase contrast, which requires ultra-thin specimens, limiting the choice of experiments.

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

Silicon nitride membranes can be used for windows of environmental chambers for in situ electron microscopy. We report that aberration corrected scanning transmission electron microscopy (STEM) achieved atomic resolution on gold nanoparticles placed on both sides of a 50-nm-thick silicon nitride membrane at 200 keV electron beam energy. Spatial frequencies of 1/1.2 Å were visible for a beam semi-angle of 26.5 mrad. Imaging though a 100-nm-thick membrane was also tested. The achieved imaging contrast was evaluated using Monte Carlo simulations of the STEM imaging of a sample of with a representative geometry and composition.