Automated calculations for computing the sample-limited spatial resolution in (scanning) transmission electron microscopy.

MATLAB scripts were designed to compute the sample-limited spatial resolution in transmission electron microscopy (TEM) and scanning TEM (STEM) as a function of different microscopy parameters including the electron dose eD, sample geometry, and materials parameters. The scripts can be used to select the optimum microscopy modality and optimize the experimental conditions to achieve the best possible resolution considering the limitations set by both the electron optics and the examined sample. The resolution can be computed as function of the objective opening semi-angle α for TEM and detector opening semi-angle ß for STEM. Optional code for computing a range over the sample thickness t or eD are provided as well, whereby the opening angle is optimized for each data point. The spatial resolution depends on the type of material of the nanoscale object (for example, gold or carbon nanoparticles), the type of matrix holding the objects (for example, water or ice), the depth of the nanoscale object inside the matrix, and eD. The optimization is consistent with the typical situation that carbon nanoparticles are best examined with TEM embedded in a thin matrix (t = 0.1 µm), while STEM is better suited for high atomic number objects such as gold nanoparticles in water, irrespective of t. The script also calculates the reduction of beam broadening in thick samples (t > 1 µm) using bright field STEM.

Environmental Liquid Cell Technique for Improved Electron Microscopic Imaging of Soft Matter in Solution.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 µm. We describe a variation of the conventional approach, wherein the Si3N4 windows are separated by a 10-µm-thick spacer, thus providing room for gas flow inside the liquid specimen enclosure. Adjusting the pressure and flow speed of humid air inside this environmental liquid cell (ELC) creates a stable liquid layer of controllable thickness on the bottom window, thus facilitating high-resolution observations of low mass-thickness contrast objects at low electron doses. We demonstrate controllable liquid thicknesses in the range 160 ± 34 to 340 ± 71 nm resulting in corresponding edge resolutions of 0.8 ± 0.06 to 1.7 ± 0.8 nm as measured for immersed gold nanoparticles. Liquid layer thickness 40 ± 8 nm allowed imaging of low-contrast polystyrene particles. Hydration effects in the ELC have been studied using poly-N-isopropylacrylamide nanogels with a silica core. Therefore, ELC can be a suitable tool for in situ investigations of liquid specimens.

Human γS-Crystallin-Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage.

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.