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.