Photoswitching Reagent for Super‐Resolution Fluorescence Microscopy

Author:

Go Ga‐eun1,Jeong Uidon1,Park Hyunbum1,Go Seokran1,Kim Doory12ORCID

Affiliation:

1. Department of Chemistry Hanyang University Seoul 04763 Republic of Korea

2. Research Institute for Convergence of Basic Science Institute of Nano Science and Technology, and Research Institute for Natural Sciences Seoul 04763 Republic of Korea

Abstract

AbstractSingle‐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 (kon and koff) 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.

Funder

Korea Toray Science Foundation

Publisher

Wiley

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