Fluorescence Lifetime Imaging Microscopy for Quantitative Characterization of Subtle Heterogeneity in Marine Bivalves...

Fluorescence lifetime imaging microscopy (FLIM) provides quantitative characterization of biochemical microenvironments, while its application in environmental biomonitoring remains limited. Here, we assess fluorescence lifetime as a parameter for resolving microenvironmental heterogeneity in a marine biomonitoring organism. Using digestive gland tissues of the marine mussel Mytilus galloprovincialis as a sentinel tissue for environmental exposure, we compared conventional histology, semiquantitative morphometric analysis, and FLIM under controlled stress conditions. Conventional histological and morphometric assessments indicated largely preserved tissue architecture across treatments. FLIM revealed significant variations in fluorescence lifetime parameters that enabled discrimination of subtle tissue differences. Analysis of lifetime parameters showed that the mean lifetime and the ratio between lifetime components were the primary contributors to analytical discrimination, whereas amplitude ratios provided limited additional information. Importantly, region-resolved analysis based on phasor plots revealed distinct lifetime signatures between different tissue compartments. FLIM detects subtle alterations beyond the resolution of morphology and complements conventional histology by enabling functional characterization at the tissue level. Together, these results establish fluorescence lifetime parameters as quantitative analytical descriptors for characterizing subtle tissue responses in environmental biomonitoring.