Biofilm formation is a complex process involving various signaling pathways and changes in gene expression. Many of the sensory mechanisms and regulatory cascades involved have been defined for biofilms formed by diverse organisms attached to solid surfaces. By comparison, our knowledge of the basic mechanisms underlying the formation of biofilms at air-liquid interfaces, i.e. pellicles, is much less complete. In particular, the roles of flagella have been studied in multiple solid-surface biofilm models, but remain largely undefined for pellicles. In this work, we characterize the contributions of flagellum-based motility, chemotaxis and oxygen sensing to pellicle formation in the Gram-positive Bacillus subtilis. We confirm that flagellum-based motility is involved in, but is not absolutely essential for, B. subtilis pellicle formation. Further, we show that flagellum-based motility, chemotaxis, and oxygen sensing are important for successful competition during B. subtilis pellicle formation. We report that flagellum-based motility similarly contributes to pellicle formation and fitness in competition assays in the Gram-negative Pseudomonas aeruginosa. Time-lapse imaging of static liquid cultures demonstrates that in both B. subtilis and P. aeruginosa, a turbulent flow forms in the tube and a zone of clearing appears below the air-liquid interface just before the formation of the pellicle, but only in strains that have flagella.

Established laser-based ionization experiments require the surface of a sample to be as flat as possible to guarantee optimal laser focus. A laser ablation electrospray ionization (LAESI) source was custom-built to accommodate the topography of non-flat sample surfaces. Employing a confocal distance sensor, a height profile of the surface in question was recorded prior to the actual ionization experiment. The robustness of the system was evaluated by the metabolic profiling of radish (Raphanus sativus) leaves, chosen due to their pronounced surface features and known content of specialized metabolites. After the ionization experiments, light microscopy imaging was performed to evaluate ablation crater size and position. Reproducible ablation mark diameters of 69 7 mm in average have been achieved. Mass spectrometric imaging capability has been proven on R. sativus leaf samples as well

This short review aims to summarize the current developments and applications of mass spectrometry-based methods for in situ profiling and imaging of plants with minimal or no sample pre-treatment or manipulation. Infrared-laser ablation electrospray ionization and UV-laser desorption/ionization methods are reviewed. The underlying mechanisms of the ionization techniques–namely, laser ablation of biological samples and electrospray ionization–as well as variations of the LAESI ion source for specific targets of interest are described.

Spatially resolving the relative distribution of analyte molecules in biological matter holds great promise in the life sciences. Mass spectrometry imaging (MSI) is a technique that can provide such spatial resolution but remains underused in fields such as chemical ecology, as traditional MSI sample preparation is often chemically or morphologically invasive. Laser ablation electrospray ionization (LAESI)-MSI is a variation of MSI particularly well-suited for situations where chemical sample preparation is too invasive but provides new challenges related to the repeatability of measurement outcomes. We assess the repeatability of LAESI-MSI by sampling a droplet of [ring- 13 C 6 ] l -phenylalanine with known concentration and expressing the resulting variability as a coefficient of variation, c v . In doing so, we entirely eliminate variability caused by surface morphology or underlying true sample gradients. We determine the limit of detection (LOD) for 13 C 6 -Phe by sampling from droplets with successively decreasing but known concentration. We assess the influence of source geometry on the LOD and repeatability by performing these experiments using four distinct variations of sources: one commercial and three custom-built ones. Finally, we extend our study to leaf and stem samples Arabidopsis thaliana and Gossypium hirsutum . We overcome the challenges of LAESI associated with three-dimensional surface morphology by relying on work previously published. Our measurements on both controlled standard and realistic samples give strong evidence that LAESI-MSI’s repeatability in current implementations is insufficient for MSI in chemical ecology.