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The recent advances in super-resolution fluorescence microscopy, including single-molecule localization microscopy (SMLM), has enabled the study of previously inaccessible details, such as the organization of proteins within cellular compartments and even nanostructures in nonbiological nanomaterials, such as the polymers and semiconductors. With such developments, the need for the development of various computational nanostructure analysis methods for SMLM images is also increasing; however, this has been limited to protein cluster analysis. In this study, we developed an edge structure analysis method for pointillistic SMLM images based on the line edge roughness and power spectral density analyses. By investigating the effect of point properties in SMLM images, such as the size, density, and localization precision on the roughness measurement, we successfully demonstrated this analysis method for experimental SMLM images of actual samples, including the semiconductor line patterns, cytoskeletal elements, and cell membranes. This systematic investigation of the effect of each localization rendering parameter on edge roughness measurement provides a range for the optimal rendering parameters that preserve the relevant nanoscale structure of interest. These new methods are expected to expand our understanding of the targets by providing valuable insights into edge nanoscale structures that have not been previously obtained quantitatively.

This article, titled "Inside Back Cover: Photoswitching Reagent for Super-Resolution Fluorescence Microscopy," discusses the photoswitching mechanism used in single-molecule localization microscopy, a popular super-resolution microscopy technique. The authors, Ga-eun Go, Uidon Jeong, Hyunbum Park, Seokran Go, and Doory Kim, propose that the rate of switch-off in photoswitching is determined by the nucleophilicity of the photoswitching reagent, while the rate of switch-on is determined by the bond dissociation energy of the photoswitching reagent-fluorophore pair. This research provides valuable insights into the understanding and improvement of super-resolution microscopy techniques. [Extracted from the article]

This article, titled "Innenrücktitelbild: Photoswitching Reagent for Super-Resolution Fluorescence Microscopy," discusses the photoswitching mechanism used in single-molecule localization microscopy, a popular super-resolution microscopy technique. The authors, Ga-eun Go, Uidon Jeong, Hyunbum Park, Seokran Go, and Doory Kim, propose that the rate of switch-off in photoswitching is determined by the nucleophilicity of the photoswitching reagent, while the rate of switch-on is determined by the bond dissociation energy of the photoswitching reagent-fluorophore pair. This research provides valuable insights into the understanding and improvement of super-resolution microscopy techniques. [Extracted from the article]

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy by exceeding the diffraction limit and revealing previously unattainable nanoscale details of cellular structures and molecular dynamics. This super-resolution imaging capability relies on fluorophore photoswitching, which is crucial for optimizing the imaging conditions and accurately determining the fluorophore positions. To understand the general on and off photoswitching mechanisms of single dye molecules, various photoswitching reagents were evaluated. Systematic measurement of the single-molecule-level fluorescence on and off rates (k on and k off ) in the presence of various photoswitching reagents and theoretical calculation of the structure of the photoswitching reagent-fluorophore pair indicated that the switch-off mechanism is mainly determined by the nucleophilicity of the photoswitching reagent, and the switch-on mechanism is a two-photon-induced dissociation process, which is related to the power of the illuminating laser and bond dissociation energy of this pair. This study contributes to a broader understanding of the molecular photoswitching mechanism in SMLM imaging and provides a basis for designing improved photoswitching reagents with potential applications extending to materials science and chemistry., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)