LAUSR

Secondary ion mass spectrometry (SIMS) is gaining popularity for molecular imaging in the life-sciences since it is label-free and allows imaging in two and three dimensions. The recent introduction of the OrbiSIMS has significantly improved the utility for biological imaging through combining sub-cellular spatial resolution with high-performance Orbitrap mass spectrometry. SIMS instruments operate in high-vacuum and samples are typically analysed in a freeze-dried state. Consequently, the molecular and structural information may not be well-preserved. We report a method for molecular imaging of biological materials, preserved in a native state, by using an OrbiSIMS instrument, equipped with cryogenic sample handling, and a high-pressure freezing protocol compatible with mass spectrometry. The performance is demonstrated by imaging a challenging sample (>90% water) of a mature Pseudomonas aeruginosa biofilm in its native state. The 3D distribution of quorum sensing signaling molecules, nucleobases and bacterial membrane molecules are revealed with high spatial-resolution and high mass-resolution. We discover that analysis in the frozen-hydrated state yields a 10,000 fold increase in signal intensity for polar molecules, such as amino acid, which has important implications for SIMS imaging of metabolites and pharmaceuticals.