biostudies-literatureBai HNIEHS NIH HHSNational Institute of General Medical SciencesNIGMS NIH HHS2793-2801https://www.ebi.ac.uk/biostudies/studies/S-EPMC8285079biostudies-literatureUnknown413(10)Three-dimensional (3D) mass spectrometry imaging (MSI) has become a growing frontier as it has the potential to provide a 3D representation of analytes in a label-free, untargeted, and chemically specific manner. The most common 3D MSI is accomplished by the reconstruction of 2D MSI from serial cryosections; however, this presents significant challenges in image alignment and registration. An alternative method would be to sequentially image a sample by consecutive ablation events to create a 3D image. In this study, we describe the use of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) in ablation-based 3D MSI for analyses of lipids within fresh frozen skin tissue. Depth resolution using different laser energy levels was explored with a confocal laser scanniAnalytical and bioanalytical chemistryThree-dimensional (3D) imaging of lipids in skin tissues with infrared matrix-assisted laser desorption electrospray ionization (MALDESI) mass spectrometry.PMC8285079R01 GM087964R01GM087964P30 ES025128Muddiman DCLinder KEBai HfalseThree-dimensional (3D) imaging of lipids in skin tissues with infrared matrix-assisted laser desorption electrospray ionization (MALDESI) mass spectrometry.Three-dimensional (3D) mass spectrometry imaging (MSI) has become a growing frontier as it has the potential to provide a 3D representation of analytes in a label-free, untargeted, and chemically specific manner. The most common 3D MSI is accomplished by the reconstruction of 2D MSI from serial cryosections; however, this presents significant challenges in image alignment and registration. An alternative method would be to sequentially image a sample by consecutive ablation events to create a 3D image. In this study, we describe the use of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) in ablation-based 3D MSI for analyses of lipids within fresh frozen skin tissue. Depth resolution using different laser energy levels was explored with a confocal laser scanni2021-01-01T00:00:00Z2021 Apr2025-04-04T20:16:22.262Z2025-04-04T20:16:22.262ZS-EPMC82850793338884710.1007/s00216-020-03105-6