Analysis of immune synapses by τau-STED imaging and 3D-quantitative colocalization of lytic granule markers

Over the last decades, intensive research studies have been focused on describing how the immunological synapse is formed, the intracellular mechanisms that control lytic granules formation, and even further, the steps toward granule polarization before the killing event is achieved. These convoluted processes pose significant experimental challenges since the components' sizes are smaller than the diffraction limit of the conventional fluorescent microscopy techniques and their highly dynamic nature. Here, we describe a procedure to perform a quantitative analysis of the protein markers of these lytic granules by using τau-STED imaging and 3D-quantitative colocalization of lytic granule markers. The innovative technology offered by τau-STED microscopy and unbiased imaging analysis is a great tool that could be applied to further our understanding of lytic granule composition and localization and study other dynamic processes at the immunological synapses.