MAss Spectrometry imaging Data Analysis in EYE – MASDA-EYE

By combining the advantages of mass spectrometry (MS) and microscopy in a single experiment, the emerging mass spectrometry imaging (MSI) technology brings extraordinarily powerful capabilities to understand the molecular complexity found in both health and disease. Compared to traditional immunocytochemistry, which is often limited by the specificity of the applied labels and the number of compounds that may be studied simultaneously, MSI gives access to molecular information on hundreds of chemical or biological molecules in a single experiment without requirement of a priori knowledge of the target compounds. In recent years, MSI has become of major interest for many clinical and biological applications, including discovery of biomarkers that display interesting spatial localization in neurodegenerative diseases or muscular dystrophies, biodistribution of drugs and metabolites, analysis of pathogens, and classification of tumor biopsies. Matrix-assisted laser desorption ionization (MALDI) and secondary ions mass spectrometry (SIMS) are two major techniques allowing desorption/ionization of molecules for subsequent time-of-flight (TOF). These technologies allow to image complementary classes of biomolecules depending on their mass (peptides/proteins, metabolites, lipids), with specific spatial resolutions (50 to 0.4 µm). While MALDI-TOF and TOF-SIMS MSI have been used separately so far, our project aims at taking advantage of their complementarity by using both techniques in parallel, on the highest-performance devices, and merging the data. In addition to being technologically challenging, the objective of merging the two image modalities highly depends on the development of advanced algorithms and software tools for data handling (gigabytes of data volumes in specific formats), processing (baseline correction, mass calibration) and interpretation (hierarchical clustering, blind positive-source separation). In particular, innovative marker identification tools are essential for MSI, since protein identification is currently hampered by the complex mixture of compounds, and lipid and metabolite databases are at their infancy. As a proof of principle, we will validate this approach in the biomedical context of ophthalmology. Glaucoma is a severe ocular disease leading to blindness and requiring lifelong treatment. Over the past 15 years, there has been accumulating scientific evidence concerning the chronic toxicity preservatives (e.g. Benzalkonium chloride, BAC) contained in antiglaucomatous eye drops. The biological aim of the study will be to understand the spatial distribution of BAC before investigating the physiopathological consequences (apoptosis, oxidative stress, inflammatory process) in a rabbit model and in human. Our approach of combining MALDI-TOF and TOF-SIMS imaging modalities with optimized software tools, and its validation for the pharmaco-toxicology of the eye, should pave the way for "histolomics", i.e. functional high-throughput molecular histology integrating complementary imaging modalities.