In Situ Real‐Time Imaging of Benzyl‐Naphthalimide Dyes Revealing Diverse Structural Evolution Dynamics at the Single‐Assembly Level

Abstract Fluorescence imaging has emerged as a powerful technique for in situ monitoring of self‐assembly dynamics providing critical insights into structural evolution and functional potential. However, the inherent complexity of supramolecular systems presents significant challenges for real‐time visualization, particularly in capturing the growth kinetics of individual assemblies and the hierarchical formation of network structures at the single‐assembly level. Here, we visualized the dynamic growth and network formation of benzyl‐naphthalimide dye assemblies in situ by confocal laser scanning microscopy (CLSM). In situ time‐lapse imaging revealed distinct dynamic growth mechanisms among the three assemblies. Theoretical calculations demonstrated that different amino substituents significantly influenced assembly morphologies by modulating the planarity of the naphthalene ring. Furthermore, fluorescence lifetime imaging revealed pronounced intra‐assembly heterogeneity, highlighting diverse molecular packing arrangements within individual assemblies. The confocal in situ visualization technique provided single‐assembly growth dynamics, offering insights unattainable through conventional spectroscopic methods. Our work reveals that naphthalimide‐based molecules can self‐assemble into a variety of distinct architectures, underscoring their potential for designing advanced functional materials with tunable properties.