Characterization of Fluorophore-Quencher Pairs for Distance-Dependent Molecular Sensing in Photoacoustic Imaging

Photoacoustic imaging coupled with molecularly responsive sensors is emerging as an attractive alternative to traditional fluorescence sensors in the deep-tissue quantification of specific molecular analytes. The range of fluorophore/quencher pairs suitable for this approach remains limited. In response, here we have systematically evaluated a set of near-infrared fluorophores for their ability to support photoacoustic imaging coupled with molecularly responsive sensors. Specifically, we have used a DNA molecular ruler to precisely vary the fluorophore-quencher distance while monitoring the absorbance, fluorescence, and photoacoustic spectra of five near-infrared fluorophores. Our results confirm previous findings identifying IRDye 800CW as a strong candidate and further establish ICG and IRDye 800 as highly promising fluorophores for responsive photoacoustic applications. We also demonstrated robust tissue penetration and preserved distance-dependent behavior under biologically relevant conditions, with ICG achieving the highest signal-to-noise ratio and the most predictable quenching response. These findings expand the palette of near-infrared probes and provide a clear framework for designing next-generation photoacoustic molecular sensors for in vivo imaging.