We have successfully established a new preparation method for 3D mass imaging of biological materials with a thermal film-based laser microdissection sysytem. This system enables to enhance ionization of hydrophobic peptides (or to restrain ionization of many molecules in biologizal mixure) on MALDI/TOF, so as to detect a small amount of peptides such as amyloid beta from frozen brain sections. The laser microdissection can capture size from 5 to 150 μm in diameter, suggesting that a single cell MS imaging (even subcellular MS imaging) is available. Using this system we prepared 3D mass image of amiloid beta peptide in hippocampus of 25 weeks-old APP-PS1 transgenic mouse and found that monomer and dimer of amiloid beta peptide are differentially located in hippocamus. Our system can also apply for mass imaging of LC/MS analysis by extracting or processing with chemically or enzymatically treatment of individual micro-dissected tissues on the thermal film. It is useful to investigate distribution of drugs or chemicals in tissue samples as a single cell size.