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Life on the nanoscale has been made accessible in recent decades by the development of fast and noninvasive techniques. High-speed atomic force microscopy (HS-AFM) is one such technique that shed light on single protein dynamics. Extending HS-AFM to effortlessly incorporate mechanical property mapping while maintaining fast imaging speed allows a look deeper than topography and reveal details of nanoscale mechanisms that govern life. Here, we present high-speed in-line force mapping (HS-iFM) to record mechanical properties and topography maps with high spatiotemporal resolution. Using HS-iFM, a comprehensive study of the nanoscale mechanical properties of living Escherichia coli revealed localized stiffening and details during cell division, formation and diffusion of pores in the membrane, and the impact of depressurization of a cell. The frame time was as low as 15 seconds with a spatial resolution of 5.5 nanometers per pixel in topography and 22 nanometers per pixel in force maps, allowing the capture of transient phenomena on bacterial surfaces in striking detail.

Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation. Here, we visualize the translocation, via one unit of a SecYEG-SecA-embedded nanodisc, of an unfolded substrate protein by high-speed atomic force microscopy (HS-AFM). Additionally, the uniform unidirectional distribution of nanodiscs on a mica substrate enables the HS-AFM image data analysis, revealing dynamic structural changes in the polypeptide-crosslinking domain of SecA between wide-open and closed states depending on nucleotides. The nanodisc-AFM approach will allow us to execute detailed analyses of Sec proteins as well as visualize nano-scale events of other membrane proteins., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

In the previous study (Green Chem., 2023, 25, 3418), highly stretchable and mechanically tough poly(methyl acrylate) (pMA) microparticle-based elastomers can be formed by drying a microparticle-containing aqueous dispersion. This discovery has the potential to overcome the mechanical weakness of industrially produced aqueous latex films. However, in 3D-arranged particle films, structural complexity, such as the existence of defects, makes it difficult to clearly understand the relationship between the particle film structure and its mechanical properties. In this study, 2D-ordered pMA particle monolayers at the air/water interface of a Langmuir trough are prepared. Under high compression at the air/water interface, the microparticles contact their neighboring particles, and the resulting monolayers can be successfully transferred onto a solid substrate. The compression of the monolayer films is linked to an increase in the elastic modulus of the monolayer film on the solid substrate as evident from the local Young's modulus mapping using atomic force microscopy. Thus, pMA particle films with different mechanical properties can be created using a Langmuir trough., (© 2024 The Author(s). Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)