The diffraction limit of visible light governs the spatial resolution of optical microscopy to roughly half the wavelength of the light, typically around 200 nm in the lateral dimension and 500 nm in the axial dimension. However, many subcellular structures and processes occur on a much smaller length scale. Technological solutions beyond the optical diffraction limit are needed to study subcellular organizations of proteins and their related functions. Super-resolution microscopy (SRM) represents a family of techniques capable of resolving structures and objects below the optical diffraction limit. This chapter provides an overview of three super-resolution techniques, stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM), and their applications in investigating subcellular protein organizations and related functions in immune cells.