The Z-sections of images acquired by confocal microscopy were imported into IMARIS software for 3D surface rendering and quantitative analysis. Surfaces were generated by selecting the source channel and setting the smooth surface detail to 0.2 μm. Background subtraction was set to 0.6 μm. The threshold for surfaces was adjusted to ensure complete coverage of all voxels and the surface area and volumes of the generated surfaces for both channels were quantified with IMARIS. The overlap between the 3D surface-reconstructed images (for ASC and SUMO1 or ZBTBT16 and ASC) was processed using the IMARIS surface-surface overlap module. This module tracked the overlapping surfaces and calculated the areas of surface colocalization. Data was exported to Excel for further analysis.
