MALDI-guided SIMS: Multiscale Imaging of Metabolites in Bacterial Biofilms

Abstract

Mass spectrometry imaging (MSI) is a versatile tool for visualizing molecular distributions in complex biological specimens, but locating microscopic chemical features of interest can be challenging in samples that lack a well-defined anatomy. To address this issue, we developed a correlated imaging approach that begins with performing matrix-assisted laser desorption/ionization (MALDI) MSI to obtain low-resolution molecular maps of a sample. The resulting maps are then used to direct subsequent microscopic secondary ion mass spectrometry (SIMS) imaging and tandem mass spectrometry (MS/MS) experiments to examine selected chemical regions of interest. By employing MALDI undersampling, the sample surface is left mostly unperturbed and available for the SIMS analysis, while also generating an ablation array that can be used for navigation in SIMS. We validated this MALDI-guided SIMS approach using cultured biofilms of the opportunistic pathogen Pseudomonas aeruginosa; bioactive secondary metabolites, including rhamnolipids and quinolones, were detected and visualized on both macro- and microscopic size scales. MSI mass assignments were confirmed with in situ MALDI MS/MS and capillary electrophoresis–electrospray ionization MS/MS analysis of biofilm extracts. Two strains of P. aeruginosa were compared, wild type and a quorum sensing mutant, and differences in metabolite abundance and distribution were observed.

Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms

“…PQS and AQNOs subclasses produce specific CRM features (Fig. S1) enabling their differentiation (16,31).…”

“…We utilized two chemically information rich imaging approaches, secondary ion mass spectrometry (SIMS) and confocal Raman microscopy (CRM) (31)(32)(33), to analyze the relative abundance and spatial distribution of AQs within 2D intact P. aeruginosa swarm communities both with and without antibiotic challenges. In the absence of antibiotics, our in situ observations of P. aeruginosa swarms revealed that the PQS and AQNO subclasses act on different spatial scales within plateassay pre-biofilm swarm communities.…”

Metal-assisted polyatomic SIMS and laser desorption/ionization for enhanced small molecule imaging of bacterial biofilms

“…jsweedle@illinois.edu assortment of sample preparation strategies, such as the application of metal films and nanoparticles, [15][16][17][18] or the use of patterned surfaces and clathrate nanostructures, 19,20 among other approaches. 21,22 Requiring no sample preparation beyond what is necessary for high vacuum compatibility, secondary ion mass spectrometry (SIMS) offers a small molecule imaging approach that is complementary to MALDI.…”

“…23,25 On the other end of SIMS inquiry, researchers have focused on developing novel sample treatment strategies broadly aimed at improving ionization. Innovations have included exposure to reactive vapor, 26 and application of traditional MALDI matrices, 27,28 metal films, 16,29 or nanoparticles. 30 Applying a thin layer of gold or silver to the sample is particularly appealing due to the wide availability of conventional sputter-coating devices for electron microscopy.…”

“…44 We have previously developed methods for the analysis of biofilms using both MetA-LDI and monatomic (Au þ primary ion beam) MetA-SIMS, and found that coating the biofilm surface with a few nanometers of gold improved sensitivity for lipids and small molecule secondary metabolites in LDI and monatomic SIMS. 16 In separate work, we created methods that correlate C 60 -SIMS imaging with confocal Raman microscopy, and applied them to image several small molecule secondary metabolites in the same biofilm model. 5,27 In the present study, we have found that while C 60 -SIMS, MetA-C 60 -SIMS, and MetA-LDI are all applicable for imaging small molecules in P. aeruginosa biofilms, each method presents its own unique advantages.…”