We developed a method for analysis and imaging of biological samples in their native state, by combining a cryo-OrbiSIMS instrument with cryogenic sample handling and high-pressure freezing. By using this method, we did analysis and imaging of frozen-hydrated mature Pseudomonas aeruginosa biofilm, which allows the identification and map of quorum sensing signaling molecules, nucleobases and bacterial membrane molecules with high spatial-resolution and high mass-resolution. Some of quorum sensing signaling molecules were further confirmed by MS/MS. By comparing the analysis of frozen-hydrated Pseudomonas aeruginosa biofilm with the freeze-dried one, we dicover that signal intensity of all interesting molecules get enhanced in the frozen-hydrated state. Especially for polar molecules, such as amino acid, it could even achieve 10,000 fold increasing. Here, we provide the original OrbiSIMS data including MS and MS/MS spectra, depth profile and images of frozen-hydrated and freeze-dried Pseudomonas aeruginosa biofilm. The data could be open by using SurfaceLab Version 7.0 (ION-TOF, Germany).

This work has received funding from the MetVBadBugs project of the EMPIR programme co-financed by the Participating States and from the European Union's Horizon 2020 research and innovation programme.