Mass spectrometry imaging is one of the fast growing technological science that can unveil fine details of molecular distributions directly on tissues. In the last nine years, major focus of our group has been developing high-resolution mass spectrometry imaging technology to study plant metabolism at single cell or subcellular level. We have now achieved laser spot size below five micron and can easily adjust last spot size for various size. We have successfully applied this high-resolution chemical imaging to visualize cellular level heterogeneity of lipids and metabolites in maize leaves and roots. The lack of chromatographic separation is a critical limitation in mass spectrometry imaging to confidently identify metabolites. To overcome this limitation, we have developed multiplex mass spectrometry imaging technique that acquires high-resolution mass spectra and tandem mass spectra directly on the tissue in a single data acquisition. We have further expanded this technique to include polarity switching so that both positive and negative ion mass spectrometric images can be acquired, then furthermore advanced to enable mass spectrometry imaging in metabolomics scale using multiple matrices on consecutive tissue sections. Lack of available matrices is one of the major limitations in matrix-assisted laser desorption ionization (MALDI)-based mass spectrometry imaging. We have performed a large scale nanoparticle screening to enhance the metabolic coverage. Also discussed is the future perspectives of this technology, including mass spectrometry imaging of isotopomers (i.e., imaging of metabolite flux) and on-tissue chemical modification to improve sensitivity.