Spatial stable isotope tracing metabolic imaging is a cutting-edge technique designed to investigate tissue-specific metabolic functions and heterogeneity. Traditional matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) techniques often struggle with low coverage of low-molecular-weight (LMW) metabolites, which are often crucial for spatial metabolic studies. To address this, we developed a high-coverage spatial isotope tracing metabolic method that incorporates optimized matrix selection, sample preparation protocols, and enhanced post-ionization (MALDI2) techniques. We employed this approach to mouse kidney, brain, and breast tumors to visualize the spatial dynamics of metabolic flow. Our results revealed diverse regional distributions of nine labeled intermediates derived from 13C6-glucose across glycolysis, glycogen metabolism, and the tricarboxylic acid (TCA) cycle in kidney tissues. In brain sections, we successfully mapped six intermediates from the TCA cycle and glutamate-glutamine (Glu-Gln) cycle simultaneously in distinct neurological regions. Furthermore, in breast cancer tumor tissues, our approach facilitated the mapping of nine metabolic intermediates in multiple pathways, including glycolysis, the pentose phosphate pathway (PPP), and the TCA cycle, illustrating metabolic heterogeneity within the tumor microenvironment. This methodology enhances metabolite coverage, enabling more comprehensive imaging of isotope-labeled metabolites and opening new avenues for exploring the metabolic landscape in various biological contexts.