The interplay between mitochondria and lipid droplets (LDs) plays a central role in regulating the β-oxidation and storage of fatty acids (FA) and is also engaged in responding to external stimuli such as nutrient deficiency. However, a single fluorescent probe enabling the discriminative and simultaneous visualization of the two organelles has not been reported yet, which brings limitation for the in-depth study on their interplay. In this work, utilizing the intramolecular spirocyclization reaction of rhodamine dyes that can dramatically change the optical and soluble properties, we have designed a new single fluorescent probe for labeling LDs and mitochondria in clearly separated dual-emission channels. The newly designed “biform” probe, MT-LD, presented in a ring-opened form in mitochondria to give a strong red emission, while it underwent the intramolecular spirocyclization reaction to target LDs showing an intense blue fluorescence. In this manner, MT-LD can label LDs and mitochondria in blue and red fluorescence, respectively. With this robust probe, the increase of mitochondria–LD contact and peridroplet mitochondria (PDM) amount during oleic acid treatment and starvation-induced autophagy has been successfully revealed. The interaction between the two organelles was also visualized in different tissues, which revealed an obviously higher level of mitochondria–LD contact and PDM amount in brown adipose tissue and lung tissue. This work provides a promising molecular tool to investigate the interplay between mitochondria and LDs and promotes studies on FA metabolism and autophagy.

The interactions between different organelles are ubiquitous and crucial for life activities. Thus, development of a single fluorescent probe enabling the simultaneous two-color visualization of two organelles is of great significance for the study of organelle interplay. Herein, using the reversible ringopening/closing reactions of rhodamine dyes, we have fabricated a robust fluorescent probe to distinguish lipid droplets (LDs) and the endoplasmic reticulum (ER) in dual-emission channels with negligible crosstalk. The probe 6′-which was sensitive to the changes in the water content in the organism, displayed strong green fluorescence in the hydrophobic LDs from its ring-closed form, while it existed in a ring-opened form in the ER to illuminate a strong near-infrared emission. Importantly, the spectral difference was up to 320 nm, and thus the crosstalk between two channels was negligible. With the unique probe, the lipid accumulation in cells treated with different concentrations of oleic acid, cholesterol, and stearic acid has been successfully observed. The changes of LDs and the ER in living cells stimulated by temperature changes and hypoxia stimulation have also been revealed. Meanwhile, the different sizes and distribution of LDs and the ER in various tissues were also studied using the robust probe. This work provides a new approach to the design of dual-emissive probes and contributes to a significant molecular tool to promote the study of organelle interactions.

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Mitochondrial membrane potential (ΔΨ m ) is an important biophysical parameter playing central roles in cell apoptosis, mitochondrial dysfunction, and other biological and pathological processes. Herein, we have rationally designed and fabricated a unique fluorescent probe for convenient ΔΨ m visualization based on hot-band absorption and controllable anti-Stokes shift emission. The robust probe was excitable via hot-band absorption and emitted anti-Stokes upconversion emission and Stokes downconversion fluorescence simultaneously. The anti-Stokes emission could be efficiently inhibited upon the binding to RNA. The cationic probe targeted mitochondria in living cells with high ΔΨ m and displayed both anti-Stokes green emission and ordinary red fluorescence. After the decrease of ΔΨ m , the probe immigrated out of mitochondria to RNA and nucleolus, which showed only red emission owing to the inhibition of anti-Stokes fluorescence. In this manner, the ΔΨ m could be visualized in dual-color mode. The probe enabled clearly monitoring the reversible changes in ΔΨ m and was successfully employed to visualize oxidative damage of living cells. The decrease of ΔΨ m in living tissues was also successfully observed with the newly designed probe.

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