Integrated Single-Cell Mass Spectrometry Imaging and Microscopy for Multimodal Analysis of Immune Cells

Understanding cellular heterogeneity within complex biological systems is essential for uncovering mechanisms of health and disease. Spatial and multimodal single-cell technologies offer powerful opportunities to link molecular phenotypes to cellular morphology and microenvironmental context, yet lipid and metabolic profiling at singlecell resolution remains technically challenging. In particular, mass spectrometry imaging (MSI)-based lipid analysis is often limited by batch-related variability, that can obscure biological signals, resolution constraints as well as insufficient co-registration with complementary modalities. 
Here, we present two approaches that address these challenges: First, an integrated multimodal workflow that combines matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with immunofluorescence microscopy to enable lipid, surface protein, and morphological profiling of the same cells. We further implemented a strategy to reduce batch effects in single cell MSI data, allowing comparisons across different samples and experimental conditions. Applying this approach to human immune cells in clinical samples from patients with liver cirrhosis, we resolve pronounced heterogeneity in lipid profiles that correlates with protein expression, morphology and maturation status. Second, we developed a transmission
mode MALDI ion-source that integrates in-source bright-field and fluorescence microscopy, allowing for (sub-)cellular investigation in both modalities with inherent coregistration. This, in combination with dedicated immunofluorescence labeling, allowed for the investigation of lipid profiles of tumor infiltrating immune cells correlated to their individual microenvironments.
Together, this work establishes multimodal single-cell methods that integrates spatial lipid analyses with fluorescence-based phenotyping while addressing key technical limitations of MSI. The approach provides a powerful tool for cell biology and translational research, enabling the discovery of disease-relevant cellular states within readily available patient samples in the form of blood, as well as in complex tissues.