Intermediate states of molecular self-assembly from liquid-cell electron microscopy

Abstract

Traditional single-molecule methods do not report whole-molecule kinetic conformations, and their adaptive shape changes during the process of self-assembly. Here, using graphene liquid-cell electron microscopy with electrons of low energy at low dose, we show that this approach resolves the time dependence of conformational adaptations of macromolecules for times up to minutes, the resolution determined by motion blurring, with DNA as the test case. Single-stranded DNA molecules are observed in real time as they hybridize near the solid surface to form double-stranded helices; we contrast molecules the same length but differing in base-pair microstructure (random, blocky, and palindromic hairpin) whose key difference is that random sequences possess only one stable final state, but the others offer metastable intermediate structures. Hybridization is observed to couple with enhanced translational mobility and torsion-induced rotation of the molecule. Prevalent transient loops are observed in error-correction processes. Transient melting and other failed encounters are observed in the competitive binding of multiple single-stranded molecules. Among the intermediate states reported here, some were predicted but not observed previously, and the high incidence of looping and enhanced mobility come as surprises. The error-producing mechanisms, failed encounters, and transient intermediate states would not be easily resolved by traditional single-molecule methods. The methods generalize to visualize motions and interactions of other organic macromolecules.

Probing Thermoresponsive Polymerization-Induced Self-Assembly with Variable-Temperature Liquid-Cell Transmission Electron Microscopy

“…[21][22][23][24][25][26] Specifically, real-time monitoring by LCTEM has been shown to provide mechanistic information regarding morphological evolution in soft matter. 27,28 For example, Patterson and coworkers were able to observe the formation of vesicles from poly(ethylene oxide)-block-poly(ε-caprolactone) via a solvent-switch approach inside the liquid-cell transmission electron microscope. 29 Time-resolved in situ imaging revealed that vesicles were nucleated by liquid-liquid phase separation, specifically by polymer-rich liquid droplets in solution.…”

Emergence and Rearrangement of Dynamic Supramolecular Aggregates Visualized by Interferometric Scattering Microscopy

“… 9 , 10 High-speed atomic force microscopy (AFM) enables the visualization of dynamic behavior during molecular self-assembly, however it has limited temporal resolution and has to be performed on surfaces, so is unsuitable for solution measurements. 11 Transmission electron microscopy (TEM), cryogenic (cryo-TEM) or liquid cell (LCTEM) transmission electron microscopy, can provide information about the structure and the morphology of the aggregates, 12 but the preparation of samples involves drying, staining, 13 or cryogenic vitrification steps 14 and the collection of images often leads to radiation damage to the sample. 15 , 16 In addition, these techniques require careful data interpretation, as well as availability of complex instrumentation.…”

Recent Progress in Using Graphene as an Ultrathin Transparent Support for Transmission Electron Microscopy

“…GLCs have also facilitated exciting research and findings in the field of nanoscience, such as crystallization and in situ crystal growth, [ 182 ] nucleation and shape formation of crystals, [ 183 ] study of molecular structure of water and ice, [ 184 ] lithium transfer in battery materials, [ 185 ] high‐resolution study of beam‐sensitive biological cells, [ 186 ] study of intermediate states during the self‐assembly of DNA double helices, [ 187 ] and exceptional spatial resolution study of nanocrystal etching on a single particle level. [ 188 ]…”