O. J. R. Gustafsson, T. M. Guinan, D. Rudd, H. Kobus, K. Benkendorff, N. H. Voelcker

Nanostructure-based mass spectrometry imaging (MSI) is a promising technology for molecular imaging of small molecules, without the complex chemical background typically encountered in matrix-assisted molecular imaging approaches.Here, we have enhanced these surfaces with silver (Ag) to provide a second tier of MSI data from a single sample. MSI data was acquired through the application of laser desorption/ionization mass spectrometry to biological samples imprinted onto desorption/ionisation on silicon (DIOS) substrates. Following initial analysis, ultra-thin Ag layers were overlaid onto the samples followed by MSI analysis (Ag-DIOS MSI). This approach was first demonstrated for fingermark small molecules including environmental contaminants and sebum components. Subsequently, this bimodal method was translated to lipids and metabolites in fore-stomach sections from a 6-bromoisatin chemopreventative murine mouse model. DIOS MSI allowed mapping ofcommon ions in fingermarks as well as 6-bromoisatin metabolites and lipids in murine fore-stomach. Furthermore, DIOS MSI was complemented by the theAg-DIOS MSI ofAg-adductable lipids such as wax esters in fingermarks and cholesterol in murine forestomach.Gastrointestinal acid condensation products of 6-bromoisatin, such as the 6,6'-dibromoindirubin mapped herein, are very challenging to isolate and characterize. By re-analysing the same tissue imprints,this metabolite was readily detected by DIOS,placed in a tissue-specific spatial context, and subsequently overlaid with additional lipiddistributions acquired using Ag-DIOS MSI. The ability to place metabolite and lipid classes in a tissue-specific context makes this novel method suited to MSIanalyses where the collection of additional information from the same sample maximises resource use,andalso maximises the number of annotated small molecules, in particular for metabolites that are typically undetectable with traditional platforms.