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.
We present two mitochondria-immobilized fluorescent probes ECPI-12 and IVPI-12 for long-term mitochondria visualization and tracking regardless of MMP changes.
The feedback from mitochondrial membrane potential (MMP) in different situations (normal, decreasing, and vanishing) can reflect different cellular status, which can be applied in biomedical research and diagnosis of the related diseases. Thus, the efficient and convenient detection for MMP in different situations is particularly important, yet the operations of current fluorescent probes are complex. In order to address this concern, we presented herein a spatially dependent fluorescent probe composed of organic cationic salt. The experimental results from normal and immortalized cells showed that it could accumulate in mitochondria selectively when MMP was normal. Also, it would move into the nucleus from mitochondria gradually with the decrease of MMP, and finally it targeted the nucleus exclusively when MMP vanished. According to the cell morphology, there is a straightforward spatial boundary between the nucleus and cytoplasm where mitochondria locate; thus, the three situations of MMP can be point-to-point indicated just by fluorescence images of the probe: that all probes accumulate in mitochondria corresponds to normal MMP; that probes locate both in the mitochondria and nucleus corresponds to decreasing MMP; that probes only target the nucleus corresponds to vanishing MMP. It is worth noting that counterstaining results with S-11348 indicated that the spatially dependent probe could be applied to distinguishing dead from viable cells in the same cell population. Compared with the commercial Cellstain-Double staining kit containing calcein-AM and propidium iodide (PI), this probe can address this concern by itself and shorten the testing time, which brings enormous convenience for relevant researches.
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