High-resolution multicolor imaging of mitochondria in lymphocytes

The identification of cellular changes that accompany immune activation has been a long-standing interest for immunologists. Among these, alterations in the metabolic states of these cells have gained particular attention in the last decade due to the emergence of the field of immunometabolism. A thorough investigation of these metabolic changes can only be achieved with an in-depth visualization of mitochondrial organization; however, current strategies for mitochondrial imaging have been optimized in model cells with a high cytoplasm-to-nucleus ratio and thus are not readily adaptable for many immune cells. Here, we devised a multicolor high-resolution microscopy strategy to image mitochondrial morphology in lymphocytes at both their resting and activated states. Our method allowed us to stain both the mitochondrial surface (by targeting TOM-20) and the mitochondrial matrix (through the use of Mitotracker dyes) while efficiently excluding nonviable cells. Our novel imaging strategy offers a powerful tool to study changes in mitochondrial morphology and complements any research focusing on lymphocyte metabolism.