Fig. 3: Quantification of liver tissue ¹³C levels using MIMS-EM.

A, B Correlated ¹³C-to-¹²C (¹³C/¹²C) ratiometric images acquired using MIMS and registered to scanning electron microscopy (SEM) of hepatocytes to create MIMS-EM maps. Data from mice continuously infused with [U-¹³C]-glucose at 40 mg/min/kg of [U-¹³C]-glucose for 1 or 4 hours. C Quantification of ¹³C/¹²C ratios in the biomass of mice shown in A, B. Data from n = 3 animals per time point. Each dot represents the mean ¹³C enrichment in a ~40 um² liver section. D, E MIMS ¹³C/¹²C ratiometric images registered to hepatocyte SEM micrographs to create MIMS-EM maps. Data from mice continuously infused with15 or 40 mg/min/kg of total body mass for 2 hours. F Quantification of ¹³C/¹²C ratios in the biomass of mice from shown in D, E). In A, B and D, E, magenta and orange colors represent lower and higher different levels of ¹³C enrichment, respectively. Dotted magenta line marks the terrestrial background for ¹³C. Data from n = 3 animals per time point. Each dot represents the mean ¹³C enrichment in a ~40 um² liver section. G Cartoon illustration showing the single cell analysis of MIMS-EM data. Figure created using biorender.com. H Representative hepatocyte SEM, ¹³C/¹²C ratio, and overlay of several organelle segmentation masks, listed on the right. Data from a mouse continuously infused with 40 mg/min/kg of [U-¹³C]-glucose for 4 hours. I ¹³C/¹²C ratios of individual hepatocytes from mice continuously infused with 40 mg/min/kg of [U-¹³C]-glucose for 1 or 4 hours. Each color represents data from a different animal. J ¹³C/¹²C levels by type of organelle after 1 or 4 hours of 40 mg/min/kg [U-¹³C]-glucose infusion. K Relative fraction of hepatocyte cell area occupied by endoplasmic reticulum (ER), lipid droplets (LDs), glycogen, mitochondria, or nucleus. Data from n = 3-to-7 mice per group, from overnight fasted mice or from mice continuously infused with 40 mg/min/kg of [U-¹³C]-glucose for 1 or 4 hours. In I, J, data from n = 6-to-9 mice per group. In C and F), a One-way ANOVA with a two-stage Benjamini, Krieger, and Yekuteli test was used, and p-values < 0.001 are shown. In J, K), One-way ANOVA with Dunns test post-hoc where ***p < 0.001. In I–K, each dot represents a single cell. In (C and F-K), data shown as 95% of the confidence interval (C.I.). In (A), scale bar 2 microns; in D and H, 5 microns.