Super-resolution microscopy has revolutionized the field of cellular imaging by overcoming the diffraction limit of conventional light microscopy and enabling the visualization of cellular structures in the nanometre range. In this protocol, a detailed procedure for super-resolution imaging of fixed primary murine T cells is described, focusing on preparation, staining, and post-processing. Imaging is performed using a super-resolution stimulated emission depletion (STED) microscope with a confocal laser scanning unit. After imaging, the data is processed using deconvolution and segmentation to reconstruct high-resolution images that enable detailed analysis of the T cell structures. This method enables a deeper understanding of T cell biology by revealing the intricate details of their internal organization and interactions.