Fig. 3. B.A./NAD(P)H FLIM resolves age-dependent changes to mitochondria across and within tissues.

A,B Schematic of imaging locations throughout the C. elegans body (A) and time points (B; blue dashed lines) imaged for the data presented in Fig. 3. B shows a lifespan of WT (N2) C. elegans (n = 100 C. elegans). C-D) WT B.A./NAD(P)H average photon arrival time images and analysis of C. elegans in germline, PM, BWM, and hypodermal cells in young (Day 1) or old (Day 11) C. elegans (n = 5–32 C. elegans per condition; moderated t test; median). E LASSO regression models generated with B.A./NAD(P)H FLIM endpoints from either PM, BWM, or hypodermal mitochondria individually or altogether. Plots depict age predicted by the models (Mito-NAD(P)H Age) versus chronological age. The blue lines indicate when predicted age hypothetically matches chronologic age. Each dot represents one C. elegans. RMSE stands for root mean square error (n = 13–32 C. elegans per condition; Student’s t test). F Correlation matrix between B.A./NAD(P)H lifetime in PM, BWM, and hypodermal (hyp) cells in individual animals across all ages analyzed in WT C. elegans showing the overall extent to which B.A./NAD(P)H FLIM lifetimes (Ƭm) across cell types in the same animal correlate with each other throughout aging. G Plot of BWM versus hypodermal B.A./NAD(P)H lifetime colored by age. Each dot represents an individual C. elegans with each axis reporting the B.A./NAD(P)H lifetime for each tissue respectively within the same C. elegans. Green lines mark the edge of the young lifetime distribution (n = 124 C. elegans). H Plot of anterior versus posterior BWM B.A./NAD(P)H lifetime (Ƭm) colored by age. Each dot represents an individual C. elegans with each axis reporting the B.A./NAD(P)H lifetime (Ƭm) for each location in the BWM respectively within the same C. elegans. Green lines mark the edge of the young lifetime distribution (n = 131 C. elegans). Scale bars, 10 µm. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.