Fig. 5: Automated single-cell analysis of lipids in their histological tissue context.
From: Spatial biology using single-cell mass spectrometry imaging and integrated microscopy
a Annotation of an H&E stain of a 4T1 tumor section; ROI of the t-MALDI-2-MSI conducted on a consecutive section marked in blue. b FM using Hoechst 33342 (nuclei, DAPI channel, blue), CellMask Green Actin Tracking Stain (actin, FITC channel, gray), IF stains for neutrophil cells (Ly6G, TxRed channel, red), immune cells (CD45, Cy5 channel, yellow), and T3 neutrophils (DcTRAIL-R1, Cy7 channel, not pictured in (b)) prior to MALDI matrix application. c t-MALDI-2-MSI overlays in positive ion mode of acylcarnitine [ACAR 18:0 + H]+ (m/z 428.38, yellow), [PC(O 32:0) + H]+ (m/z 720.59, magenta), [PC(34:1) + H]+ (m/z 760.58, cyan) and [TG 52:4 + H]+ (m/z 855.75, blue) corroborating histological annotations from (a), (b). d Zoom-in of blue squares (b) and (c). FM shows Hoechst (blue), CellMask (gray), neutrophils (red) and immune cells (yellow). MSI overlays: [cholesterol-H2O + H]+ (m/z 369.35, yellow), [PC(O-32:0) + H]+ (m/z 720.59, magenta), [LPC(16:0) + H]+ (m/z 496.34, cyan). e Zoom-in of red squares in (b) and (c). FM shows neutrophils (red), immune cells (yellow) and T3 neutrophils (cyan). MSI overlays: [ACAR 18:0 + H]+ (m/z 428.38, yellow), [PC(O-32:0) + H]+ (m/z 720.59, magenta) and [PC(O-38:5) + H]+ (m/z 794.61, cyan). f Zoom-in of green squares in (b) and (c). FM shows Hoechst (blue), CellMask (gray), neutrophils (red) and immune cells (yellow). MSI overlays: Molecular ion of Heme B (m/z 616.18, yellow), [PC(O-32:0) + H]+ (m/z 720.59, magenta) and [SM(34:1;O2) + H]+ (m/z 703.57, cyan). g 63,408 cell masks based on the FM image inside the complete ROI. Zooms show cell masks overlaid with FM and MSI. For MSI ion signal intensity of [PC(O 32:0) + H]+ at m/z 720.59 selected exemplarily.
