High Resolution Imaging of Nonequilibrium Colloidal Self-Assembly via Photofixation

The self-organization of colloidal nanoparticles into complex structures, both in equilibrium and out-of-equilibrium, is a growing area in colloidal science with potential for creating functional materials. While equilibrium assemblies form stable and periodic structures, out-of-equilibrium (or active) assemblies exhibit dynamic, reconfigurable behavior under external stimuli. Therefore, understanding the structure–function relationships in these assemblies remains challenging due to their transient nature and limitations of current characterization methods. In this work, we present a methodology termed Fixation and Resolving of Colloidal Active Matter Ensembles (FRAME). FRAME combines UV photopolymerization to fix nonequilibrium colloidal assemblies with high-resolution imaging techniques, including 3D confocal microscopy, SEM and 3D STED super-resolution imaging, for subsequent structural characterization. We applied this method to Optical Matter (OM) structures formed within an optical trap at the glass/water interface. Using FRAME, we conducted a detailed analysis of OM structures composed of colloidal nanoparticles ranging from 200 nm to 1 μm. We demonstrate the robustness of this method by validating that the fixation process does not alter structural properties, allowing for accurate structural analysis. FRAME offers a distinct approach for investigating nonequilibrium colloidal assemblies, enabling the way for their rational design and application across a broad range of colloidal systems.