Comparative Analysis of Fluorophore-MALDI Matrix Interactions in Solid and Liquid Phases to Investigate Fluorescence Enhancement Effects in FluoMALDI Imaging

Fluorescence microscopy and matrix-assisted laser desorption/ionization (MALDI) imaging are important spatial biology tools which drive biomedical research. We recently developed the FluoMALDI pipeline [1], which integrates slide-scanning fluorescence microscopy (SSFM) with MALDI imaging. FluoMALDI is based on the discovery that MALDI matrix co-crystallization with fluorophores significantly enhances fluorescence intensity, which can be leveraged in fluorescence applications with low fluorescence signal. Here we investigated the effects of MALDI matrix-fluorophore interactions on fluorescence enhancements in liquid and solid states. Various fluorophores including Rhodamine B (Rhod), Alexa dyes (A488, A555, A647), and Protoporphyrin IX (PPIX) were tested with commonly used MALDI matrices including α-Cyano-4-hydroxycinnamic acid (CHCA), 9-Aminoacridine (9AA), Norharmane (nH), and 2,5-dihydroxybenzoic acid (DHB). In solid-phase experiments, dried fluorophore droplets on indium-tin-oxide (ITO) microscopy slides were robotically sprayed with several passes of MALDI matrix, followed by SSFM measurement at various excitation/emission wavelengths. For liquid-phase experiments, we measured the excitation and emission spectra of various fluorophore-MALDI matrix mixtures at different ratios in 3:2 acetonitrile/water in a fluorescence spectrometer. In solid phase, we observed that increasing the matrix density by doubling the layers of robotically sprayed matrix onto dried PPIX, A555, and other dried fluorophore droplets significantly enhanced their fluorescence signals for particular matrices. Most notably we observed fluorescence enhancement when combining, CHCA or DHB with A488, nH or CHCA with A555, and 9AA with PPIX. In liquid phase, higher matrix concentrations did not further enhance the fluorescence signal; however, increasing the dye concentrations led to slight fluorescence signal enhancements. In ongoing work, we are investigating other fluorophore-matrix pairs in solid and liquid phases.