Measurement raw data, experimental descriptions, and protocols created during the MSCA-PF-01 101067953 MASS2 - SALDI-SPICI-MSI. Measurements and optimization of Au-based surface-assisted laser desorption/ionization mass spectrometry imaging combined with single-photon induced chemical ionization. The project aimed to develop a novel means for extensive metabolome and lipidome analysis by SALDI-PI-MSI to overcome limitations of MALDI. For this,we combined of a new PI strategy coined Single-Photon Induced Chemical Ionisation (SPICI) with functionalised Au layers. With this, the chemical coverage of compounds in biomedical tissue sections could be extended, especially for an extended mass range below 500 Da, and with high mass and spatial resolution. The parameter were optimised and key settings for a comprehensive analysis identified. As a result, a large number of endogenous analytes were detected that could not be detected with conventional MALDI-MSI or SALDI-MSI systems before. Complex data outputs were analyzed with the versatile in-house developed software package rMSI that facilitated routine data mining. The new technical possibilities were demonstrated in the analysis and comprehensive spatial biochemical characterisation of zebra fish embryos. This can now serve as a fast and scalable template for, e.g., future toxicology/exposome studies.