Optical metabolic imaging identifies metabolic shifts and mitochondria heterogeneity in POLG mutator macrophages

The polymerase gamma (POLG) gene mutation is associated with mitochondria and metabolism disorders, resulting in heterogeneous responses to immunological activation and posing challenges for mitochondrial disease therapy. Optical metabolic imaging captures the autofluorescent signal of two coenzymes, NADH and FAD, and offers a label-free approach to detect cellular metabolic phenotypes, track mitochondria morphology, and quantify metabolic heterogeneity. In this study, fluorescence lifetime imaging (FLIM) of NAD(P)H and FAD revealed that POLG mutator macrophages exhibit a decreased NAD(P)H lifetime, and optical redox ratio compared to the wild-type macrophages, indicating an increased dependence on glycolysis. FLIM revealed that both wild-type and POLG mutator macrophages switch to a decreased NAD(P)H τ ₁ , and τ _m after immune stimulation by Lipopolysaccharides (LPS). Furthermore, a bimodality index of subpopulation analysis identified heterogenous populations of POLG mutator macrophage responses under immune challenge by LPS. Moreover, to quantify the mitochondria variations in POLG mutator macrophages, a customized thresholding image processing pipeline was developed to segment mitochondria regions within each cell from the NADH image, allowing for the feature analysis of mitochondria clusters. Consequently, the wild-type macrophages exhibited a higher percentage of mitochondria-containing pixels and longer lengths of connected mitochondria, as compared with POLG mutated macrophages. Altogether, these results illustrate the potential of optical metabolic imaging for non-invasive detection and quantification of cellular metabolism, metabolic heterogeneity within cell populations, and intra-cellular mitochondria morphology differences in POLG mutator macrophages. Optical metabolic imaging will be valuable for studying POLG-mutation diseases and evaluating efficacy of potential therapies.