A novel FRET-based fluorescent probe capable of simultaneously imaging lipid droplets and the endoplasmic reticulum with two distinct fluorescence signals in HeLa cells

Inter-organellar interactions play indispensable roles in regulating cellular homeostasis, necessitating advanced methodologies for their simultaneous and discriminative visualization. Fluorescent probes, prized for their sensitivity and spatiotemporal resolution, are pivotal tools for elucidating organelle dynamics in live-cell studies. However, current technologies remain limited in achieving robust dual-color imaging of multiple organelles with minimal crosstalk. To address this gap, we developed a Förster resonance energy transfer (FRET)-based ratiometric probe leveraging the pH-responsive spiro-pyran motif, which undergoes reversible ring-opening/closing transitions. This probe enables concurrent dual-color visualization of lipid droplets (LDs) and the endoplasmic reticulum (ER) in HeLa cells under single-excitation conditions, achieving high Pearson’s correlation coefficients and minimal spectral overlap. Our work advances the design of multifunctional probes for decoding inter-organelle communication in live systems.