Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue

Fluorescence lifetime (FLT) multiplexing and multispectral imaging (MSI) are both frequently employed for in vitro and ex vivo biological studies. In vivo applications of MSI for deep seated fluorophores require consideration of diffusive light propagation in biological tissue. We have previously shown that a well-known redshift of fluorescence spectra in diffusive medium induces a fluorophore cross-talk, which cannot be accounted for even with known optical properties of the medium. In contrast, FLT measurements remain largely unaffected by light propagation in tissue, enabling zero cross-talk and accurate relative quantification. While a fully quantitative estimation of fluorophore concentrations requires depth resolved tomographic imaging, this is often not possible due to the difficulty of estimating tissue optical properties and modelling light propagation in complex tissue geometries. Here, we experimentally investigate the performance of planar (non-tomographic) MSI and FLT multiplexing for the quantitative recovery of multiple near-infrared fluorophores embedded in 4-8 mm thick tissue. We show that FLT multiplexing provides a superior quantification accuracy (error < 10%) compared to MSI (error = 20-107%) in tissue. The error rates for MSI increased with tissue thickness and can be directly attributed to the spectral redshift induced cross-talk between emission spectra. Our data indicate that planar FLT multiplexing can provide high quantification accuracy in thick biological tissue without a need for optical property estimation, thereby offering an important validation tool for rapid quantification of fluorophore concentrations in bulk tissue.