A single fluorescent probe for dual-color imaging and polarity precise analysis in lipid droplets and endoplasmic reticulum.

Background: Elaborating the subcellular polarity is pivotal for pathophysiological research and early disease diagnosis. Despite its significance, achieving simultaneous two-color fluorescence imaging and quantitative analysis of polarity across multiple organelles remains challenging. This limitation primarily stems from the lack of single fluorescent (SF) probes capable of such precise and multifaceted functionality.

Results: We introduce a novel SF probe LE-TPA, designed for concurrent dual-color imaging and precise polarity determination in lipid droplets (LDs) and the endoplasmic reticulum (ER) under a single excitation. LE-TPA adopts a D-π-A-π-D configuration, which demonstrates highly selective and sensitive fluorescence response to polarity variations in Oleic acid-THF or THF-water mixtures, accompanied with an exceptional linearity (R > 0.99) between the max λ and polarity parameter Δf, paving the way for quantitative polarity analysis in live samples. Furthermore, LE-TPA enables simultaneous, real-time imaging of these organelles due to their different water contents, and provides compelling evidence for supporting the hypothesis that LDs derive from the ER. Importantly, LE-TPA effectively identifies polarity differences between healthy and cancerous cells at the subcellular level and allows precise polarity mapping of non-alcoholic fatty liver disease (NAFLD) tissues at different pathological stages.

Significance: These findings highlight the versatility of probe LE-TPA as a powerful tool for subcellular polarity studies and related disease diagnosis.