MALDI IMS-Derived Molecular Contour Maps: Augmenting Histology Whole-Slide Images

Abstract

Imaging mass spectrometry (IMS) provides untargeted, highly multiplexed maps of molecular distributions in tissue. Ion images are routinely presented as heatmaps and can be overlaid onto complementary microscopy images that provide greater context. However, heatmaps use transparency blending to visualize both images, obscuring subtle quantitative differences and distribution gradients. Here, we developed a contour mapping approach that combines information from IMS ion intensity distributions with that of stained microscopy. As a case study, we applied this approach to imaging data from Staphylococcus aureus-infected murine kidney. In a univariate, or single molecular species, use-case of the contour map representation of IMS data, certain lipids colocalizing with regions of infection were selected using Pearson's correlation coefficient. Contour maps of these lipids overlaid with stained microscopy showed enhanced visualization of lipid distributions and spatial gradients in and around the bacterial abscess as compared to traditional heatmaps. The full IMS data set comprising hundreds of individual ion images was then grouped into a smaller subset of representative patterns using non-negative matrix factorization (NMF). Contour maps of these multivariate NMF images revealed distinct molecular profiles of the major abscesses and surrounding immune response. This contour mapping workflow also enabled a molecular visualization of the transition zone at the host-pathogen interface, providing potential clues about the spatial molecular dynamics beyond what histological staining alone provides. In summary, we developed a new IMS-based contour mapping approach to augment classical stained microscopy images, providing an enhanced and more interpretable visualization of IMS-microscopy multimodal molecular imaging data sets.

Figure 1.

Annotated murine kidney. (A) Autofluorescence microscopy image of a murine kidney. Fluorescently labeled S. aureus can be seen by the green fluorescence. (B) QuPath software was used to perform threshold-based segmentation of fluorescently labeled S. aureus, shown in yellow. (C) Pathologist-annotated regions of the kidney (renal pelvis, renal medulla, renal cortex, adrenal gland) and regions pertaining to infection (inflammatory cell infiltrate, major abscess).

Figure 2.

Contour map of a single MALDI IMS ion image correlating to a staphylococcal abscess. (A) Periodic acid-Schiff (PAS) stain of an S. aureus abscess within the renal cortex of a murine kidney. White areas in the tissue show freeze artifacts resulting from tears. (B) Image of [PC(O-32:0) + Na]⁺ (m/z 742.572, −0.09 ppm) that was found to colocalize with regions of staphylococcal infection based on a pixelwise Pearson correlation analysis. (C) Contour map generated using the ion image for [PC(O-32:0) + Na]⁺, with contours labeled by m/z intensity values. (D) Contour map overlaid with PAS.

Figure 3.

NMF components of IMS data. Non-negative matrix factorization (NMF) was performed on the IMS data, and each of the resultant components were visualized as heat maps. (A) NMF component corresponding with the renal medulla. (B) NMF component corresponding to the renal cortex. (C) NMF component corresponding to the inflammation surrounding the major abscess. (D) NMF component corresponding to the inflammatory cell infiltrate.

Figure 4.

Contour maps built upon results of multivariate NMF analysis. Non-negative matrix factorization (NMF) was applied to the MALDI IMS data, discerning 13 notable molecular patterns. (A) Contour map of NMF component 5, which correlates to the lipids surrounding inflammatory response. Contours are labeled by intensity values. (B) Contour map of an NMF component 6, which correlates to the lipids in the bacterial abscess. Contours are labeled by intensity values. (C) Contour map of NMF component 5 overlaid with PAS. (D) Contour map of NMF component 6 overlaid with PAS. (E) Average spectra corresponding to each of the NMF components, revealing distinct molecular profiles between the surrounding inflammation (top) and bacterial abscess (bottom).

Figure 5.

Three-dimensional contour map based on results of multivariate NMF analysis. (A) 3D contour map of NMF component 6, correlating with the bacterial abscess. (B) 3D contour map of NMF component 5, correlating with the inflammatory response.