In living systems, subcellular organelles mutually cooperate and closely contact to form organelle interaction networks. Thus, the simultaneous and discriminative visualization of different organelles is extremely valuable for elucidating their distribution and interplay. However, such meaningful investigations remain a great challenge due to the lack of advanced single fluorescent probes (SFprobes) capable of simultaneous and two-color imaging of two targets. Herein, for the first time, we present two excited-state intramolecular proton transfer (ESIPT) based SF-probes (PPC and EPC) for simultaneous two-color fluorescence imaging of lipid droplets (LDs) and the endoplasmic reticulum (ER) under single-wavelength excitation. Due to the strong electron-donating ability of the side substituents, the fluorescence spectra and colors of these ESIPT probes are highly sensitive to the nuance of water contents between LDs and ER, leading to orange and green fluorescence in LDs and ER, respectively, in the Lambda imaging mode. Using the probe PPC or EPC, the morphology, size, and distribution of LDs and ER have been investigated in live cells and tissues. With the aid of in situ and real-time fluorescence imaging in Lambda mode, we observed the generation of newborn LDs near the ER regions and their close apposition and shared identical fluorescence colors, probably providing a valuable proof for the mainstream hypothesis that LDs originate from the ER. The remarkable imaging performances render these SF-probes as powerful tools to decipher LD-ER related biological processes.

Lipid droplets are highly associated with obesity, diabetes, inflammatory disorders and cancer. A reliable two-photon dye for specific lipid droplets imaging in live cells and live tissues at ultra-low concentration has rarely been reported. In this work, four new aggregation-induced emission luminogens (AIEgens) based on the naphthalene core were designed and synthesized for specific two-photon lipid droplets staining. The new molecules, namely NAP AIEgens, exhibit large Stokes shift (>110 nm), high solid-state fluorescence quantum yield (up to 30%), good two-photon absorption cross section (45-100 GM at 860 nm), high biocompatibility and good photostability. They could specifically stain lipid droplets at ultra-low concentration (50 nM) in a short time of 15 min. Such ultra-low concentration is the lowest value for lipid droplets staining in live cells reported so far. In vitro and ex vivo two-photon imaging of lipid droplets in live cells and live mice liver tissues were successfully demonstrated. In addition, selective visualization of lipid droplets in live mice liver tissues could be achieved at a depth of about 70 μm. These excellent properties render them as promising candidates for investigating lipid droplets-associated physiological and pathological processes in live biological samples.

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