Unraveling Ferroptosis Mechanisms: Tracking Cellular Viscosity with Small Molecular Fluorescent Probes.

Ferroptosis is a recently identified form of regulated cell death characterized by iron accumulation and lipid peroxidation. Numerous functions for ferroptosis have been identified in physiological as well as pathological processes, most notably in the treatment of cancer. The intricate balance of redox homeostasis is profoundly altered during ferroptosis, leading to alteration in cellular microenvironment. One such microenvironment is viscosity among others such as pH, polarity, and temperature. Therefore, understanding the dynamics of ferroptosis associated viscosity levels within organelles is crucial. To date, there are a very few reviews that detects ferroptosis assessing reactive species. In this review, we have summarized organelle's specific fluorescent probes that detects dynamics of microviscosity during ferroptosis. Also, we offer the readers an insight of their design strategy, photophysics and associated bioimaging concluding with the future perspective and challenges in the related field.

Ratiometric fluorescent probes for pH mapping in cellular organelles.

The intracellular pH (pHi) in organelles, including mitochondria, endoplasmic reticulum, lysosomes, and nuclei, differs from the cytoplasmic pH, and thus maintaining the pH of these organelles is crucial for cellular homeostasis. Alterations in the intracellular pH (ΔpHi) in organelles lead to the disruption of cell proliferation, ion transportation, cellular homeostasis, and even cell death. Hence, accurately mapping the pH of organelles is crucial. Accordingly, the development of fluorescence imaging probes for targeting specific organelles and monitoring their dynamics at the molecular level has become the forefront of research in the last three decades. Among them, ratiometric fluorescent probes minimize the interference from the excitation wavelength of light, auto-fluorescence from probe concentration, environmental fluctuations, and instrument sensitivity through self-correction compared to monochromatic fluorescent probes, which are known as turn-on/off fluorescent probes. Small-molecular ratiometric fluorescent probes for detecting ΔpHi are challenging yet demanding. To date, sixty-two ratiometric pH probes have been reported for monitoring internal pH alterations in cellular organelles. However, a critical review on organelle-specific ratiometric probes for pH mapping is still lacking. Thus, in the present review, we report the most recent advances in ratiometric pH probes and the previous data on the role of mapping the ΔpHi of cellular organelles. The development strategy, including ratiometric fluorescence with one reference signal (RFRS) and ratiometric fluorescence with two reversible signals (RFRvS), is systematically illustrated. Finally, we emphasize the major challenges in developing ratiometric probes that merit further research in the future.

A Ratiometric Fluorescent Probe for Hypochlorite and Lipid Droplets to Monitor Oxidative Stress.

Mitochondria are valuable subcellular organelles and play crucial roles in redox signaling in living cells. Substantial evidence proved that mitochondria are one of the critical sources of reactive oxygen species (ROS), and overproduction of ROS accompanies redox imbalance and cell immunity. Among ROS, hydrogen peroxide (H2O2) is the foremost redox regulator, which reacts with chloride ions in the presence of myeloperoxidase (MPO) to generate another biogenic redox molecule, hypochlorous acid (HOCl). These highly reactive ROS are the primary cause of damage to DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins, leading to various neuronal diseases and cell death. Cellular damage, related cell death, and oxidative stress are also associated with lysosomes which act as recycling units in the cytoplasm. Hence, simultaneous monitoring of multiple organelles using simple molecular probes is an exciting area of research that is yet to be explored. Significant evidence also suggests that oxidative stress induces the accumulation of lipid droplets in cells. Hence, monitoring redox biomolecules in mitochondria and lipid droplets in cells may give a new insight into cell damage, leading to cell death and related disease progressions. Herein, we developed simple hemicyanine-based small molecular probes with a boronic acid trigger. A fluorescent probe AB that could efficiently detect mitochondrial ROS, especially HOCl, and viscosity simultaneously. When the AB probe released phenylboronic acid after reacting with ROS, the product AB-OH exhibited ratiometric emissions depending on excitation. This AB-OH nicely translocates to lysosomes and efficiently monitors the lysosomal lipid droplets. Photoluminescence and confocal fluorescence imaging analysis suggest that AB and corresponding AB-OH molecules are potential chemical probes for studying oxidative stress.