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Dual-organelle molecular localizers represent powerful new tools allowing the exploration of interorganelle physical contacts and subcellular chemical communication. Here, we describe new dynamic molecular probes to localize mitochondria and lipid droplets taking advantage of the differential proton gradients present in these organelles as well as the activity of mitochondrial esterase. We unveil their potential utility when organelle retention mechanisms and proton gradients are synchronized, an insight that has not been documented previously. Our discoveries indicate that dual-organelle probes serve as a valuable multiplexing assay during starvation-induced autophagy. The pioneering molecular mechanism they employ opens doors to avoid using labile esters such as acetoxymethyl derivatives which are not optimal in imaging microscopy assays.

This study introduces a paradigm-shifting approach to optimize mitochondrial targeting. Employing a new fluorescent probe strategy, we unravel a combined influence of both Nernst potential ( Ψ ) and partitioning ( P ) contributions. Through the synthesis of new benz[ e ]indolinium-derived probes, our findings redefine the landscape of mitochondrial localization by optimizing the efficacy of mitochondrial probe retention in primary cortical neurons undergoing normoxia and oxygen-glucose deprivation. This methodology not only advances our understanding of subcellular dynamics, but also holds promise for transformative applications in biomedical research and therapeutic development.

Lipid droplets (LDs) are intracellular organelles found in most cell types from adipocytes to cancer cells. Although recent investigations have implicated LDs in numerous diseases, the current available methods to monitor them in vertebrate models rely on static imaging using fluorescent dyes, limiting the investigation of their rapid in vivo dynamics. Here, we report a fluorophore chemistry approach to enable in vivo LD dynamic monitoring using a Nernstian partitioning mechanism. Interestingly, the effect of atorvastatin and osmotic treatments toward LDs revealed an unprecedented dynamic enhancement. Then, using a designed molecular probe with an optimized response to hydration and LD dynamics applied to Zebrafish developing pericardial and yolk-sac edema, which represents a tractable model of a human cardiovascular disease, we also provide a unique dual method to detect disease evolution and recovery.

Using intracellular-controlled photochemistry to track dynamic organelle processes is gaining attention due to its broad applications. However, most of the employed molecular probes usually require toxic photosensitizers and complex bioanalytical protocols. Here, the synthesis and performance of two new subcellular probes ( MitoT1 and MitoT2 ) are described. The probes undergo photooxidation in the damaged tissue of zebrafish, a model system for tissue regeneration studies. Using high-resolution confocal microscopy and fluorescence spectroscopy, we combine the mentioned photoinduced interconversion at the homeostatic membrane viscosity to track singlet oxygen activity selectively. The continuous and real-time biosensing method reported here provides a new approach for simultaneously detecting endogenous singlet oxygen and viscosity status.

Mitochondrial voltage dynamics plays a crucial role in cell healthy and disease. Here, a new fluorescent probe to monitor mitochondrial early voltage variations is described. The slowly permeant probe is retained in mitochondria during measurements to avoid interferences from natural membrane potential by incorporating an hydrolizable ester function. Voltage, local polarity, pH parameters and transmembrane dynamics were found to be deeply correlated opening a approach in mitochondrial sensing.

Membrane tension pores determine organelle dynamics and functions, giving rise to physical observables during the cell death process. While fluorescent organelle-targeted probes for specific chemical analytes are increasingly available, subcellular dynamic processes involving not only chemical parameters but also physicochemical and physical parameters are uncommon. Here, we report a mitochondrial chemical probe, named RCN , rationally designed to monitor osmotic effects during transmembrane tension pore formation by using local mitochondrial polarity and a subcellular localization redistribution property of the probe. Utilizing fluorescence spectroscopy, high-resolution confocal imaging, and spectrally resolved confocal microscopy, we provide a new correlation between mitochondrial dynamics and bleb vesicle formation using osmotic pressure stimuli in the cell, where the mitochondrial local polarity was found to drastically increase. The RCN provides a reliable protocol to assess transmembrane pore formation driven by osmotic pressure increments through local polarity variations and is a more robust physicochemical parameter allowing the health and decease status of the cell to be measured.

A multicomponent diversity-oriented synthesis of new highly emissive tetracyclic isoquinolines that target specific organelles is described. The title compounds were prepared via a three-step protocol starting with an Ugi four-component reaction, followed by either an intramolecular alkyne hydroarylation and subsequent alkene isomerization or through a Pomeranz-Fritsch-type cyclization with a final intramolecular Heck reaction. Subcellular localization studies of these compounds using green channel confocal microscopy revealed remarkable and distinctive distribution patterns in live cells, showing an unprecedented high selectivity and imaging contrast. The differentiated organelle visualization-including localizers for mitochondria, lysosomes, Golgi apparatus, endoplasmic reticulum, and plasma membrane-was achieved by varying the nature of the tetracyclic system and substituent pattern, changing the original four-component set in the starting Ugi reaction.

Small-molecule fluorescent probes having optimized optical properties, such as high photostability and brightness, local microenvironment sensitivity and specific subcellular localizations, are increasingly available. Although the basis for designing efficient fluorophores for bioimaging applications is well established, implementing an improvement in a given photophysical characteristic always tends to compromise another optical property. This problem has enormous consequences for in vivo imaging, where ensuring a specific localization and precise control of the probe response is challenging. Herein we discuss a fluorescent probe, CC334, as a case study of the chromenylium-cyanine family that commonly exhibits highly complex photophysical schemes and highly interfered bioanalytical responses. By an exhaustive and concise analysis of the CC334 optical responses including detailed spectroscopic calibrations, steady-state microenvironment effects, ultrafast photophysics analysis and computational studies, we elucidate a new strategy to apply the probe in the singlet oxygen reactive oxygen species (1O2-ROS) monitoring using in vitro and in vivo models. The probe provides a new avenue for designing fluorescent probes to understand the dynamic behavior of subcellular environments.

The measurement of physicochemical parameters in living cells can provide information on individual cellular organelles, helping us to understand subcellular function in health and disease. While organelle-specific chemical probes have allowed qualitative evaluation of microenvironmental variations, the simultaneous quantification of mitochondrial local microviscosity (η m ) and micropolarity (ϵ m ), along with concurrent structural variations, has remained an unmet need. Herein, we describe a new multifunctional mitochondrial probe (MMP) for simultaneous monitoring of η m and ϵ m by fluorescence lifetime and emission intensity recordings, respectively. The MMP enables highly precise measurements of η m and ϵ m in the presence of a variety of agents perturbing cellular function, and the observed changes can also be correlated with alterations in mitochondrial network morphology and motility. This strategy represents a promising tool for the analysis of subtle changes in organellar structure., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two simple and low cost 2,4-di-tert-butyl-6-[(1-hydroxycyclohexylmethylimino)methyl]phenol (L1) and 2-[{(1-hydroxycyclohexyl)methylimino}methyl]phenol (L2) Schiff base sensors exhibiting selectivity for Zn(2+) in water:methanol (95:5, v/v, 10 mM HEPES) are described. L1 and L2 display an "off-on" fluorescence effect forming the L1·Zn and L2·Zn complexes, respectively. In the case of L1·Zn, the emission response is quenched by the addition of Cu(2+) forming the respective L1·Cu complex; in spite of that, the fluorescence signal can be completely restored only by the addition of tartrate anions (C4H4O6(2-)) forming again L1·Znvia the "off-on" displacement approach. However, in the case of L2·Zn no Cu(2+) interference is observed, which is a typical problem for Zn(2+) sensors. Here we describe that a very subtle structural change in the ligand during transition from the enol-imine tautomer in L1 to the keto-enamine tautomer in L2 is enough to modulate the Zn(2+)/Cu(2+) selectivity. Also, the Zn(2+)vs. Cd(2+) discrimination for L1 and L2 is proved. Moreover, we found that the interaction between both L·Zn complexes and tartrate anions completely restored the free ligands by the ligand substitution mechanism even in a more efficient association than phosphate anions. Further, a second colorimetric response channel upon addition of Fe(2+) was observed for L1 and L2. Then, TD-DFT theoretical calculations were conducted in order to study the efficiency of the sensors to give different responses in the presence of such metal ions. Finally, the L2 sensor successfully detects Zn(2+) in Jurkat cells cultured with and without Zn(2+) enriched medium.