Fig. 3: Lipid analysis of cultured cells at sub-cellular resolving power.

a Fluorescence image of THP-1 derived macrophages recorded before MALDI matrix application with a slide scanning microscope using Hoechst 33342 (nuclei, DAPI channel, blue), CellMask Green Actin Tracking Stain (actin, FITC channel, green) and pHrodo (phagolysosomes, Cy3 channel, red). b An overlay of three different ion signal intensity distributions measured using t-MALDI-2-MSI with 1.5 × 1.5 µm² pixel size in positive ion mode of the molecular ion of Hoechst 33342 (m/z 452.23, blue), [PE(33:1) + H]⁺ (m/z 704.52, red) and [PC(34:1) + H]⁺ (m/z 760.58, green) reveals a strong cell-to-cell heterogeneity. c and d Zoom in from (a) and (b), marked in each as a blue and red square, respectively, demonstrates intracellular differences in lipid distribution. To investigate the intracellular molecular distributions related to phagocytosis, masks for the regions with pHrodo⁺ (red) and remaining cell (green) are created based on the FM image. The outlines of the masks identified by the pHrodo stain are overlayed with the MSI results to demonstrate the quality of co-registration.