A novel multicolor stimulated emission depletion (STED) microscopy strategy to visualize mitochondrial morphology in lymphocytes

Abstract

Abstract Immunometabolism is an emerging field that studies the activation-induced reprogramming required to alter immune cell function and meet new energetic demands. In-depth investigations of the metabolism-related cellular changes necessitate high-resolution visualization of mitochondria. However, current strategies for imaging mitochondria are optimized for model cells with high cytoplasm-to-nucleus ratios and are not readily adaptable for many immune cells. Here, we devised a multicolor STED microscopy method to assess B lymphocyte mitochondria in resting and activated states. By staining specimens with TOM-20 and MitoTracker dyes, as well as DAPI and a viability dye, our strategy allowed us to assess the morphology and volumes of mitochondria while efficiently excluding dead cells. Our analyses showed that stimulation of B lymphocytes via toll-like receptor 9 (TLR9) and/or B cell receptor (BCR) led to notable mass and volume increases in individual mitochondria within 24 hours. Furthermore, TLR9-stimulated cells demonstrated more tubular and connected mitochondrial networks consistent with their higher metabolic demands. In addition, collecting images of the same specimens side-by-side on AiryScan and standard confocal laser microscopes showed that STED microscopy performed significantly better in resolving changes in 3D mitochondrial morphology. However, none of the light microscopy imaging strategies could resolve activation-induced structural alterations in cristae or mitochondrial matrix. In conclusion, our novel STED-based multicolor imaging approach offers a powerful super-resolution tool to study immunometabolism and mitochondrial reorganization in lymphocytes at the whole cell level.