Schematic workflow of metabolite ratio imaging using pixel data from Bruker Scils Lab API or imzml file from any MSI data source. For Scils lab MSI data, the execution of R codes starts with connecting a local Scils lab session project in R studio. Installation of Scils lab is required for this connection. After pixel data for a feature list is generated and exported as an EXCEL csv file, Scils lab installation is not required for downstream ratio imaging and data analysis, except for the potential writing of a spot image with labels back to Scils lab. For other types of MSI data, R code execution extracts pixel data from imzml file for subsequent ratio imaging and analysis.

Pixel-by-pixel ratio image visualization reduces cryosection preparation artefacts and enables spatial metabolic discovery in COX 10 KO mouse brain (left 4 sections) compared to WT (right four sections). Panel A-C: blurry and diffused images of LPEs with ω3 (A-B) and ω6 (C) polyunsaturated fatty acyl chains; Panel D-F: sharper ratio images of ω3 fatty acid containing LPE 20:4 and LPE 22:4 to ω6 fatty acid containing LPE 22:6; Panel G: ratio image of ω6 arachidonic acid (FA 20:4) to ω3 docosahexaenoic acid (FA 22:6) is consistent with the identified abundance ratio distributions for LPE 20:4 and LPE 22:4 to LPE 22:6.

Metabolically relevant metabolite ratio pairs that probe enzymatic and metabolic pathway activity in E8.5 mouse embryo cryosections and mouse hippocampus at 10 µm spatial resolution. Panel A: annotated drawing of E 8.5 embryo tissue, derived from the section adjacent to imaged section. Panel B-H: Ratio of substrate/ product pairs of glycolysis, fatty acid and lipid metabolism that imply compartmented activities for hexokinase (B), fatty acid desaturation (C), fatty acid chain elongation (D), fatty acid composition (E-F) and phospholipase activity (G-H) in E 8.5 embryos.

Altered fatty acid and lipid metabolism probed by ratio imaging in adipose tissue from COX 10 KO vs.WT mice. COX 10 KO, left 4 sections; WT, right 3 sections. Imaging data was acquired at 80 µm spatial resolution. Panel A: Decreased abundance ratio of FA 22:6 (DHA, ω3 fatty acid) to FA 20:4 (AA, ω6 fatty acid) in COX 10 KO adipose tissue. Panel B-C: Decreased fatty acid FA 16:0 and FA 18:0 desaturation in COX 10 KO adipose. Panel D-E: Decreased desaturated fatty acid abundance ratios for lysophospolipids and phospholipids in COX 10 KO adipose tissue. Panel H-J: Elevated fatty acid synthesis inferred by free fatty acid and phospholipids ratios, considering FA 18:0 to FA 16:0 (H-I) and FA 20:1 to 18:1 (L) in COX 10 KO adipose. Panel K-L: Elevated PLA2 (K) and PLA1(L) activities implied by the elevated ratio of Lyso PA to PA in COX 10 KO adipose.

Ratio imaging uncovers novel mouse brain regions with genotype-specific and spatially resolved ratios of aspartate, glutamate and glutamine. Panel A-C: images of glutamine, glutamate and aspartate in coronal sections of COX 10 KO (left 4 sections) and WT (right 4 sections) mouse brains. Panel D-F: Differential abundance ratios of aspartate, glutamate and glutamine observed in defined regions of the COX 10 KO and WT brains. Panel G-H: Reciprocal and differential aspartate to glutamate ratio and Arc-like NAC containing region was repeated in a separate group of COX 10 KO (left 4 sections) and WT (right four sections) mouse brains. Panel I-J: Enrichment in the glutamate to aspartate ratio in the cortex region of mouse brain, as revealed in horizonal and sagittal sections.

Spearman correlation of an unknown with ratio and non-ratio entities

Brain glutamate to glutamine ratio positively correlates FeCl2 using combined ratio and non-ratio pixel data and spearman correlation

Brain glutamine to glutamate ratio negatively correlates FeCl2 using combined ratio and non-ratio pixel data and spearman correlation

PCA segmentation of mouse brains comparing ratio and non-ratio metabolite pixel data. Panel A: PCA considering the top 5 components from ratios of 10-glutamate related metabolites that reveal an unanticipated arc-like regional distribution within the hypothalamus, striatum and NAc, that is not detected by consideration of only non-ratio metabolite data. Panel B: PCA considering the top 5 components 5-LPE metabolites showing fine brain structure that is not fully represented by only non-ratio metabolite segmentation.

list of glutamate metabolism related metabolites for ratio generation and PCA segmentation of brain sections

list of lyso PEs for ratio generation and PCA segmentation of brain sections

Comparing correlation of neurotransmitter related metabolites and ratios in ROI7 of WT and KO brains

Metabolite ratios for segmentation and biomarker discovery in segmented ROIs. KO: right 4 sections; WT: left 4 sections. Panel A-C: Ratio images of homocarnosine, glutamine and aspartate in. Panel D: Enzymatic regulation and interconversions among brain glutamine, aspartate and homocarnosine. Panel E: Image of homocarnosine. Panel F: PCA with top 5 components and using ratios of homocarnosine, glutamine and aspartate shows differential ROI abundance shown in cyan blue in larger area of striatum, thalamus hypothalamus regions in KO compared to WT. Panel G: PCA with top 5 components and using homocarnosine, glutamine and aspartate pixel data show indistinguishable ROIs between genotypes.

UMAP analysis using 5 LPEs ratio followed by K-mean clustering on COX KO and WT brain sections shows a much cleaner and biologically relevant ROIs compared to those of LPE non-ratio. Panel A: UMAP clustering using ratios of 5 LPEs. Panel B: UMAP clustering using 5 LPE pixel data. The list of 5 LPEs is shown Table S3. Nine neighbors, 2 components with Euclidean distance as metric measurement of similarities were used in this UMAP analysis.

Application of pixel-by-pixel exemplary MALDI MS imaging data to metabolomics multivariate analysis using free or commercially available software package. Panel A: ROIs of COX 10 KO and WT brain outer cortex; Panel B: PLS-DA score plot showing classification of COX 10 KO and WT cortex pixels using Metaboanalyst 5.0 (https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml). Panel C: Variable importance in projection (VIP) scores of metabolites from PLS-DA shown in panel B. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in COX 10 KO and WT. Panel D: Matched pathways showing p values from the pathway enrichment analysis and pathway impact values from the pathway topology analysis. Differential metabolites imaged in cortex of COX 10 KO vs WT brain and metabolite quarries from KEGG mouse metabolic pathway were used in this analysis. Panel E: Metabolite set enrichment analysis showing enrichment ratio of metabolic pathway metabolites. Differential metabolites from cortex of COX 10 KO vs WT and the Small Molecule Pathway Database (SMPDB) were used for this analysis. The enrichment analysis resembles gene set enrichment analysis in transcriptomics. Panel F: Metabolite correlation heatmap generated from differential metabolites in cortex of COX 10 KO and WT. Metaboanalyst 5.0 was used for analyses shown in panel B-E and Agilent MassProfiler Professional 15.1 was used for analysis in shown in panel F.

Exemplary MALDI MSI images generated before (A) and after (B) missing value annotation and from reciprocal ratios (C-D). MSI Data was acquired at 80 µm using NEDC as matrix.

Ratio of glutamate to glutamine and Fe2+ abundance are co-enriched in the outer cortex of the COX10 KO (right 4 sections) and WT (left 4 sections) brain.

ROIs and UMAP scatter plots generated from non-ratio metabolite data. Panel A-I: Nine segmented ROIs generated UMAP and K-mean clustering analysis corresponding to clustered regions of 1-9. Panel J: Scatterplot of UMAP first 2 components (V1 vs V2) projecting the distribution and similarity of 9 segmented ROIs resulting in a incomplete and inconsistent ROIs among replicates. 5 LPE (Table S3) pixel data was used for UMAP analysis.

ROIs and UMAP scatter plots generated from metabolite ratio data. Panel A-I: Nine segmented ROIs generated UMAP and K-mean clustering analysis corresponding to clustered regions of 1-9. Panel J: Scatterplot of UMAP first 2 components (V1 vs V2) projecting the distribution and similarity of 9 segmented ROIs shows a distinct LPE profile between outer cortex (ROI 3) cortex (ROI 8) and ventral thalamus (ROI 5) striatum region(ROI2). Ratio of 5 LPE (Table S3) pixel data was used for UMAP.

Ratio images of NAA, NAAG and aspartate for UMAP-generated ROIs. Panel A-H: Ratio image of NAA to aspartate in 8 UMAP-segmented ROIs. Panel I-J: Ratio images of NAAG to NAA (I) and NAAG to aspartate in ROI 7 of UAMP segmented ROIs.

Images of NAAG and neurotransmitter related metabolites visualized in UMAP-generated ROI 7. Panel A: NAAG is enriched and elevated in in ROI 7–like region of COX 10 KO brain. Panel B-D: NAA,NAAG and glutamine are elevated in ROI 7 of COX 10 KO brain. Panel E: decreased level of aspartate in ROI 7 of COX10 KO; Panel F: Glutamate shows no change in ROI 7 of COX 10 KO compared to WT.

Pixel-by-pixel correlation of glutamine to glutamate ratio with a compendium of all detected metabolites and ratios exclusively in UMAP-generated ROI 7, comparing WT (A) to COX 10 KO (B) brain. Only significant correlations with a spearman correlation coefficient of greater than 0.4 were presented in the figure.

UMAP using 5LPE ratios generated ROIs consistent with brain anatomy in two separate experiments with horizonal and sagittal brain sections. Panel A-B: Overlay of nine segmented ROIs generated from UMAP and K-mean clustering analysis in horizonal hemisphere sections (A) and sagittal brain sections (B). Panel C-D: Scatterplot of UMAP first 2 components (V1 vs V2) projecting the distribution and similarity of 9 segmented ROIs in horizonal (C) and sagittal sections (D). See Table S3 for the list of 5 LPEs.