Mitochondrial functions are heavily influenced by acid–base homeostasis. Hence, elucidation of the mitochondrial pH is essential in living cells, and its alterations during pathologies is an interesting question to be addressed. Small molecular fluorescent probes are progressively applied to quantify the mitochondrial pH by fluorescence imaging. Herein, we designed a unique small molecular fluorescent probe, PM-Mor-OH, based on the lipophilic morpholine ligand-conjugated pyridinium derivative of “IndiFluors”. The morpholine-conjugated fluorescent probe usually localized the lysosome. However, herein, we observed unusual phenomena of morpholine-tagged PM-Mor-OH that localized mitochondria explicitly. The morpholine ligand also plays a pivotal role in tuning optical properties via photoinduced electron transfer (PET) during internal pH alteration (ΔpHi). In the mitophagy process, lysosomes engulf damaged mitochondria, leading to ΔpHi, which can be monitored using our probe. It exhibited “ratiometric” emission at single wavelength excitation (ex. 488) and is suitable for monitoring and quantifying the ΔpHi using confocal microscope high-resolution image analysis during mitophagy. The bathochromic emission shifts due to intramolecular charge transfer (ICT) in basic pH were well explained by the time-dependent density functional theory (TD-DFT/PCM). Similarly, the change in the emission ratio (green/red) with pH variations was also validated by the PET process. In addition, PM-Mor-OH can quantify the pH change during oxidative stress induced by rapamycin, mutant A53T α-synuclein-mediated protein misfolding stress in mitochondria, and during starvation. Rapamycin-induced mitophagy was further elucidated by the translocation of mCherry Parkin to damaged mitochondria, which well correlates with our probe. Thus, PM-Mito-OH is a valuable probe for visualizing mitophagy and can act as a suitable tool for the diagnosis of mitochondrial diseases.

Intracellular pH (pHi) in organelles, including mitochondria, endoplasmic reticulum, lysosome, and nuclei, differ from cytoplasmic pH, and maintaining the pH of those organelles is crucial for cellular homeostasis. Alteration of...

Mitochondria are the powerhouse of the cell and function at pH ~8.0. Dysfunctions of mitochondria, includes mitochondrial damage, leading to pH alteration. Hence, researchers aim to develop efficient pH probes for tracking mitochondrial pH dynamics. Herein, we developed a PETbased fluorescent probe for pH monitoring during mitochondrial dysfunctions. Three derivatives were synthesized with a variable spacer's length in pentacyclic pyridinium fluorophores (PM-C2, PM-C3, and PM-C6). An efficient electron transfers from the receptor (tertiary amine) was observed in the case of PM-C2 compared to the other two derivatives. This PET process was inhibited when tertiary amine was protonated in acidic pH. However, PM-C3 showed minimal fluorescence intensity at similar conditions and almost negligible change in case of PM-C6, suggesting poor PET process for both the derivatives. Furthermore, DFT/TD-DFT quantum chemical calculation well supported this optical phenomena and PET process. Biocompatible, photostable, and mitochondria-specific PM-C2 could monitor pH dynamics during mitochondrial damage which were engulfed by lysosome, also known as mitophagy. This mitophagy process were induced by rapamycin and starvation, which can be monitored by turn-on fluorescence enhancement. This process was further validated by tracking Parkin-protein translocation from cytoplasm to damaged mitochondria using our developed probe.

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase‘s active site for hydrolyzing the substrate. In addition, the fluorescent turn‐on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM‐OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD‐DFT calculation. Moreover, the substrate (PM‐OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image‐based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity.

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