The proper functioning of organs and tissues relies on the interaction, spatial organization, and specialization of all our cells. This study aims to enhance lipidomic analysis of lung tissues and kidney utilizing matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with fluorescence microscopy.

Integration of MALDI-MSI, which allows for spatial mapping of lipids, and immunofluorescence microscopy, which enables high-resolution imaging of biomolecules using specific molecular identification via antibodies, enables us to link specific lipid signatures with functional tissue units. Although previous approaches provided methods to combine and align both images, tools are still needed to perform enrichment analysis within each region. A key component of our research is the development of advanced R coding techniques to enhance data processing, visualization, interpretation and statistical analysis. By introducing novel algorithms and visualization tools, the data-processing can present a powerful and efficient tool for molecular identification and distribution.

Our workflow allows for the registration of a MALDI-MSI image, with multi-channel fluorescence microscopy images to identify which lipids are most prevalent at functional units of a tissue identified using immunohistochemical probes of cell markers in each functional unit. First, MALDI-MSI molecular assignments are downloaded from the open-source metabolite annotation platform METASPACE. Next, functional units were annotated in the fluorescent images by setting a threshold for each color channel. Thresholding allowed to choose the areas above a certain threshold value, thus allowing to annotate tissue functional units automatically. The MALDI-MSI together with the fluorescence images were registered in our MALDI data processing software and exported to our R-package for the analysis of omics data, ROmics Processor.

We found distinct lipid populations by performing student’s t-test comparisons of each functional unit, indicating pathways or biomarkers significant to the health of kidney and lung tissues. These tools could be further used to compare molecular composition of a healthy and diseased tissue at functional units to better understand cell relationships, molecular mechanisms and how lipids affect health, potentially providing therapeutic targets. It can be applied to other human tissues similarly.

Christopher Anderton

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