Abstract

We report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. Our approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm eliminates the notion that MALDI matrices obscure and obstruct optical microscopy approaches, allowing to image the exact same cells in tissues, free of any physical changes between fluorescence and MALDI imaging, which minimizes data registration processes. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. We showcase the capabilities of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.