## 6 Results
### Imaging Global Metabolomics in Multi-Organ Platform of the Mice  
Segmentation analysis was performed based on the acquired data using machine learning methods, and utilized IMAGEREVEL software and Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) [30; 31]. In Figure 5, a definitive metabolic compartment was clearly visualized in the thorax before and after DEX treatment in mice. Massive apoptosis found in the cortex of DEX-treated thymus were classified as a light green color in UMAP. Apoptosis-related metabolism in the thorax of the mice were prevalent on the cortex with DEX treatment. With advanced calculations using 82 fractions of UMAP technique, the spinal cord was clearly demarcated with white matter (pink) and grey matter (yellow) as a butterfly shape. Furthermore, the third compartment in grey matter was intermingled with blue colored compartments in Figure 6, and displayed a very similar pattern to the distribution of nucleus of interneurons in anterior and posterior horns of grey matter. With this calculation applied to the thymus, we have succeeded in dividing the metabolic compartment within the thymus as: (1) outer-most compartment consistent with thymus cortex (red), (2) outer medulla or junctional area often described as CMJ (blue), and (3) inner-most medulla (purple) in normal mice (Figure 7). Interestingly, there were several TUNEL-staining positive locations, mostly extra-thymic, which shared a consistent metabolic profile, expressed in green color. Most importantly, we have succeeded in categorizing CMJ from a metabolic point of view, in normal murine thymus. Figure 6: **UMAP analysis from tMSI data. ** (**a**) Segmentation images based on UMAP analysis were shown (**a,b**). Each segment calculated by UMAP was visualized in a three-dimensional space with graph layout algorithms (**c**). Color used in (**c**) is identical to (**a,b**). (**a**) control mouse, (**b**) DEX-treated mouse, (**d–g**) UMAP-profiled metabolic status of the spinal cord. Three metabolomic clusters were marked with blue (**d**), yellow (**e**), and pink (**f**). Yellow color is consistent with grey matter and pink color is consistent with white matter. Blue color looks like nucleus of various interneurons included in grey matter. Three colored images were merged on the HE staining of the spinal cord shown in (**g**). Figure 7: _Cont._  
Another point is that the metabolic heterogeneity of the thymic medulla was first visualized using the current technique, which was obviously influenced by DEX treatment. Notably, molecular profiles of the cortical thymus in normal mice had a unique molecular profile, shown in red color in Figure 6, which mostly disappeared in the DEX-treated thymus cortex and shifted toward juxtaposed posterior portion of the medulla. In normal mouse thymus medulla, metabolic heterogeneity was observed in a regular manner as two layers of CMJ and inner-most medulla. With DEX treatment, the metabolic compartment representing CMJ (blue) and the innermost medulla (purple) compartment in normal thymus was intermingled, as shown in Figure 6. These observations have a close relationship to the histological findings with TUNEL and purple (**e**). The metabolic compartment representing the  
positive cells (Figure 6) before and after DEX treatment, and the distribution of metabolites corresponding to the structure and distribution of each organ was visualized as a cluster map (Figure 6). Crucially, the aforementioned trends above were reproduced well by independent experiments, as shown in Supplementary Figure S7. Here, we have shown a novel DEX-responsive molecule, mostly depicted in the cortex of the thymus (Figure 8). We have developed a multivariate analytical algorithm based on an IMS dataset, which enabled us to find metabolic biomarkers of systemic effect of DEX administration; however, these metabolites were not referred to in the current data base, i.e., METLIN except \(m/z\) 558.04 (ADP-ribose), as shown in Supplementary Table S1.