Lipid Signatures and Inter-Cellular Heterogeneity of Naïve and Lipopolysaccharide-Stimulated Human Microglia-like Cells.

Microglia are non-neuronal cells, which reside in the central nervous system and are known to play an important role in health and disease. We investigated the lipidomic phenotypes of human naïve and stimulated microglia-like cells by atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI). With lateral resolutions between 5 and 1.5 µm pixel size, we were able to chart lipid compositions of individual cells, enabling differentiation of cell lines and stimulation conditions. This allowed us to reveal local lipid heterogeneities in naïve and lipopolysaccharide (LPS)-stimulated cells. We were able to identify individual cells with elevated triglyceride (TG) levels and could show that the number of these TG-enriched cells increased with LPS stimulation as a hallmark for a proinflammatory phenotype. Additionally, the observed local abundance alterations of specific phosphatidylinositols (PIs) indicate a cell specific regulation of the PI metabolism.

Matrix-Free High-Resolution Atmospheric-Pressure SALDI Mass Spectrometry Imaging of Biological Samples Using Nanostructured DIUTHAME Membranes.

Applications of mass spectrometry imaging (MSI), especially matrix-assisted laser desorption/ionization (MALDI) in the life sciences are becoming increasingly focused on single cell analysis. With the latest instrumental developments, pixel sizes in the micrometer range can be obtained, leading to challenges in matrix application, where imperfections or inhomogeneities in the matrix layer can lead to misinterpretation of MS images. Thereby, the application of premanufactured, homogeneous ionization-assisting devices is a promising approach. Tissue sections were investigated using a matrix-free imaging technique (Desorption Ionization Using Through-Hole Alumina Membrane, DIUTHAME) based on premanufactured nanostructured membranes to be deposited on top of a tissue section, in comparison to the spray-coating of an organic matrix in a MALDI MSI approach. Atmospheric pressure MALDI MSI ion sources were coupled to orbital trapping mass spectrometers. MS signals obtained by the different ionization techniques were annotated using accurate-mass-based database research. Compared to MALDI MSI, DIUTHAME MS images captivated with higher signal homogeneities, higher contrast and reduced background signals, while signal intensities were reduced by about one order of magnitude, independent of analyte class. DIUTHAME membranes, being applicable only on tissue sections thicker than 50 µm, were successfully used for mammal, insect and plant tissue with a high lateral resolution down to 5 µm.

Implementation of a High-Repetition-Rate Laser in an AP-SMALDI MSI System for Enhanced Measurement Performance.

Matrix-assisted laser desorption/ionization mass spectrometry imaging is a promising tool in the life sciences for obtaining spatial and chemical information from complex biological samples. State-of-the-art setups combine high mass resolution and high mass accuracy with high lateral resolution, offering untargeted insights into biochemical processes on the single-cell length scale. Despite recent technological breakthroughs, the sensitivity and acquisition speed of many setups are often in competition with achievable pixel resolutions below 25 µm. New measurement modes were developed by implementing a high-repetition-rate laser into an AP-SMALDI10 ion source, coupled to an orbital trapping mass spectrometer. These new MSI modes allow for a modular use of the new setup. We demonstrate that the system allows single cell features to be visualized in mouse brain tissue sections at a pixel resolution of 5 µm and an imaging speed of 18 pixels/s. Furthermore, the analytical sensitivity was improved in another measurement mode by applying multiple pulses of a highly focused laser beam over larger square pixels ≥25 µm edge length, increasing ion signal intensities up to 20-fold on tissue and decreasing the limit of detection by 1 order of magnitude.

Atmospheric-Pressure MALDI Mass Spectrometry Imaging at 213 nm Laser Wavelength.

First results for a new atmospheric-pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging source operating at 213 nm laser wavelength are presented. The activation of analytes in the 213 nm MALDI process at atmospheric pressure was evaluated and compared to results for 337 nm MALDI and electrospray ionization using thermometer molecules. Different sample preparation techniques for nicotinic acid, the matrix with the highest ionization efficiency at 213 nm of all tested matrices, were evaluated and optimized to obtain small crystal sizes, homogenous matrix layer sample coverage, and high ion signal gains. Mass spectrometry imaging experiments of phospholipids in mouse tissue sections in positive- and negative-ion mode with different lateral resolutions and the corresponding pre-/post-mass spectrometry imaging workflows are presented. The use of custom-made objective lenses resulted in sample ablation spot diameters of on average 2.9 µm, allowing mass spectrometry imaging experiments to be performed with 3 µm pixel size without oversampling. The ion source was coupled to an orbital trapping mass spectrometer offering high mass resolution (>100.000), high mass accuracy (≤ ±2 ppm), and high sensitivity (single pixel on-tissue tandem MS from 6.6 µm2 ablation area). The newly developed 213 nm atmospheric-pressure MALDI source combines the high mass resolution and high mass accuracy performance characteristics of orbital trapping mass spectrometers with high lateral resolution (pixel size ∼3 µm) mass spectrometry imaging.