After validation of FRAME, we can now use it to characterize our non-equilibrium optical matter system. The 3D confocal microscopy image provides detailed information on the three-dimensional structure of the assemblies which can be extracted via image processing. To achieve this, we developed an approach based on distance matrices (see Materials and Methods). Briefly, the particles are localized in 3D using Gaussian fitting, then, the distances between every possible particle pairs are subsequently calculated in an Euclidean distance matrix. After that, iterating through each layer, we detect triangles that meet specific geometric criteria of common crystal structures (e.g., equilateral triangles with angles close to 60 degrees for hexagonal packing), which can be further analyzed across layers to identify 3D structures like hexagonal prisms. This analysis enables the extraction of the unit lattice and the characterization of the 3D structure of the permanent optical matter (Figure ). We can also determine the degree of crystallinity of the structure by calculating the ratio of particles that belongs to such unit cells. Figure shows an example of OM structure formed, subsequently made permanent using FRAME and then imaged in 3D using a confocal microscope (Figure ). Figure shows a graphical representation of the 3D assembly, realized from the 3D localization positions and the known size of the particles where the color code indicates the layer in which the particle belongs. Only the particles that were found to be part of a crystalline structure are colored, representing approximately 80% of the entire assembly. The lattice arrangement follows an ABA packing pattern, with a decrease in particle number from the top surface to the bottom of the assembly. The c/a ratio refers to the ratio of the vertical distance between layers (c) to the horizontal distance between particles (a) in the hexagonal lattice, and it helps describe how the lattice is stacked and spaced in three dimensions. The c/a ratio of the unit lattice was calculated at 1.71 ± 0.15, which is slightly higher than the theoretical lattice constant for hexagonal packing (1.63). However, the packing efficiency was found to be approximately 71.6%, calculated by averaging the c/a values for the six sides, which is slightly lower than the ideal packing density (74%) of a hcp ABA structure (Figure ). Now that we can extract the 3D structure of the non-equilibrium optical matter, it is interesting to see if we can use that feedback to learn how to control the spatial arrangement of the colloidal particles, emphasizing the role of our method in the characterization and design of non-equilibrium colloidal assemblies.