Multimodal FRACTAL-MSI and Image Fusion Improves Detection and Resolution of Biomolecule Imaging

Elemental mass spectrometry imaging (MSI) of biomolecules via metal-conjugated antibodies enables highly multiplexed, in situ quantitative analyses. However, the detection of low abundance analytes by elemental MSI and the spatial resolution of the images obtained are inhibited by detector sensitivity. To overcome this constraint, we introduce FRACTAL-MSI, a signal amplification method that adapts FluoRescent signal Amplification via Cyclic staining of TArget moLecules (FRACTAL) for multimodal laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and immunofluorescence (IF) imaging by alternating fluorescent- and lanthanide-tagged secondary antibodies on the same histological section. Using NeuN as a model neuronal marker, we demonstrate 23-80× amplification of IF and LA-ICP-MS signals in fresh-frozen murine brain, FFPE tissue, and SH-SY5Y cells. Two image fusion methods that did not require machine learning were then developed that exploited true probe colocalization to generate quantitative elemental images at IF resolution. The enhanced sensitivity permitted super-resolution reconstruction of LA-ICP-MS data to obtain subcellular images with a 250 nm resolution, or greater than that of the 40× IF obtained via image fusion. We also demonstrated selective target amplification within a 4-plex elemental panel. Together, FRACTAL-MSI provides a practical, broadly accessible strategy to improve detection, and therefore the resolution, of low-abundance biomolecules.