Diphenylbutadiene Fluorescent Analogues in Sub-cellular Imaging and Monitoring Mitophagy.

A series of donor-acceptor (D-π-A) substituted diphenylbutadienes exhibiting solvatochromic emission and a large Stokes shift (100-200 nm) were designed and synthesized for distinctive organelle labelling, enabling real-time monitoring of organelle behaviour such as lysosomal dynamics, mitophagy monitoring, and stress responses. The morpholine-substituted D-A-D diphenylbutadiene (M2) was employed to investigate selective imaging of lysosomes, the uptake of damaged mitochondria through mitophagy, and monitoring lysosomal viscosity or pH changes. Other diphenylbutadiene derivatives (M1, M3, M4) selectively accumulated in lipid droplets. All the synthesized derivatives demonstrated significant uptake in 5-day post-fertilization zebrafish larvae, with M2 showing maximum uptake in the enterocyte-containing heart and intestinal regions, which include the lysosomes.

Substitution Effects on Subcellular Organelle Localization in Live-cell Imaging.

A series of D-p-A indole-containing fluorescent probes were developed followed by an investigation of their photophysical properties and compounds' suitability for subcellular imaging in living cells. We demonstrate that the preference for mitochondrial localization was lost when morpholine was substituted, resulting in the accumulation of the molecule in the lysosomes. However, interestingly, the presence of a nitro group led to their localization within the lipid droplets despite the presence of the morpholine pendant. We also showcase the probes' sensitivity to pH, the influence of added chloroquine, and the temperature response on the changes in fluorescence intensity within lysosomes. The design of the probes with strong intramolecular charge transfer and substantial Stokes shift could facilitate extensive application in various cellular lysosomal models and contribute to a better understanding of the mechanisms involved in stimuli-responsive diseases.

A multipurpose mitochondrial NIR probe for imaging ferroptosis and mitophagy.

This paper explores the use of a di-cationic fluorophore for visualizing mitochondria in live cells independent of membrane potential. Through the synthesized di-cationic fluorophore, we investigate the monitoring of viscosity, ferroptosis, stress-induced mitophagy, and lysosomal uptake of damaged mitochondria. The designed fluorophore is based on DQAsomes, cationic vesicles responsible for transporting drugs and DNA to mitochondria. The symmetric fluorophores possess two charge centres separated by an alkyl chain and are distinguished by a pyridinium group for mitochondrial selectivity, the C-12 alkyl substitution for membrane affinity, and an electron donor-π-acceptor fluorescent scaffold for intramolecular charge transfer. The synthesized fluorophores, PP and NP, emit wavelengths exceeding 600 nm, with a significant Stokes shift (130-211 nm), and NP demonstrates near-infrared emission (∼690 nm). Our study underscores the potential of these fluorophores for live-cell imaging, examining physiological responses such as viscosity and ferroptosis, and highlights their utility in investigating mitophagy damage and lysosomal uptake.

Detecting labile heme and ferroptosis through 'turn-on' fluorescence and lipid droplet localization post Fe2+ sensing.

Iron, a crucial biologically active ion essential for metabolic processes in living organisms, plays a vital role in biological functions, and imbalances in iron levels can lead to various diseases. In this study, we have developed two simple "turn-on" fluorescent probes, NOPy and NOCN, for the quick and selective detection of Fe2+ at nanomolar levels (LOD of 35 nM), accompanied by significant absorption and emission shifts, along with colorimetric demarcation. Both fluorophores exhibit an excellent "turn-on" emission response upon encountering Fe2+ in the cells. Flow cytometry and confocal fluorescence imaging studies demonstrate enhanced fluorescence signals in response to labile iron, efficiently detecting heme during erastin-induced ferroptosis. Interestingly, we also observed that the product formed after Fe2+ sensing localizes within the lipid droplets. These water-soluble and highly sensitive reactive probes, NOPy and NOCN, enable investigations of iron-dependent physiological and pathological conditions. The development of these probes represents an advancement in the field, offering a rapid and selective means for detecting Fe2+ with minimal cytotoxicity.

A two-in-one probe: imaging lipid droplets and endoplasmic reticulum in tandem.

The endoplasmic reticulum (ER) and lipid droplets (LDs) intricately interact in cellular processes, with the ER serving as a hub for lipid synthesis and LDs acting as storage organelles for lipids. Developing fluorescent probes that can simultaneously visualise the ER and LDs provides a means for real-time and specific visualisation of these subcellular organelles and elucidating their interaction. Herein, we present synthetically simple and novel donor-π-acceptor styryl fluorophores (PFC, PFN and PFB) incorporating pentafluorophenyl (PFP) to demonstrate exquisite discriminative imaging of ER and LD with a single excitation wavelength. The PFP moiety aids the ER selectivity, while the overall hydrophobicity of the molecule aids in the LD targeting. Furthermore, the fluorophores are utilised in studying the changes in size, distribution, and biogenesis of LDs within ER regions after treatment with oleic acid. Strong emission, lower concentrations ∼100 nM requirement, minimal cytotoxicity, and photostability make these fluorophores excellent tools for probing sub-cellular dynamics.

Fluorescent styrenes for mitochondrial imaging and viscosity sensing.

Fluorophores bearing cationic pendants, such as the pyridinium group, tend to preferentially accumulate in mitochondria, whereas those with pentafluorophenyl groups display a distinct affinity for the endoplasmic reticulum. In this study, we designed fluorophores incorporating pyridinium and pentafluorophenyl pendants and examined their impact on sub-cellular localization. Remarkably, the fluorophores exhibited a notable propensity for the mitochondrial membrane. Furthermore, these fluorophores revealed dual functionality by facilitating the detection of viscosity changes within the sub-cellular environment and serving as heavy-atom-free photosensitizers. With easy chemical tunability, wash-free imaging, and a favorable signal-to-noise ratio, these fluorophores are valuable tools for imaging mitochondria and investigating their cellular processes.

Lipid Droplets Specific Fluorophore for Demarcation of Normal and Diseased Tissues.

Using high-fidelity, permeable, lipophilic, and bright fluorophores for imaging lipid droplets (LDs) in tissues holds immense potential in diagnosing conditions such as diabetic or alcoholic fatty liver disease. In this work, we utilized linear and Λ-shaped polarity-sensitive fluorescent probes for imaging LDs in both cellular and tissue environments, specifically in rats with diabetic and alcoholic fatty liver disease. The fluorescent probes possess several key characteristics, including high permeability, lipophilicity, and brightness, which make them well-suited for efficient LD imaging. Notably, the probes exhibit a substantial Stokes shift, with 143 nm for DCS and 201 nm for DCN with selective targeting of the lipid droplets. Our experimental investigations successfully differentiated morphological variations between diseased and normal tissues in three distinct tissue types: liver, adipose, and small intestine. They could help provide pointers for improved detection and understanding of LD-related pathologies.

Fluorescent styryl pyridine-N-oxide probes for imaging lipid droplets.

Lipid droplets (LDs) have emerged as major regulators of cellular metabolism, encompassing lipid storage, membrane synthesis, viral replication, and protein degradation. Exclusive studies have suggested a direct link between LDs and cancer, as a notable abundance of LDs is found in cancerous cells. Therefore, monitoring the location, distribution, and movements of LDs is of paramount importance for understanding their involvement in biological processes. To target LDs, we designed and synthesized fluorophores with a styryl scaffold bearing electron-donating amino groups and pyridine-N-oxide, a zwitterionic acceptor moiety. We explored their photophysical properties in various solvents and conducted systematic DFT calculations on the synthesized fluorescent molecules, comparing them with neutral pyridine and cationic pyridinium styryl dyes. The results demonstrate that diphenylaminostyryl pyridine-N-oxide (TNO) shows excellent imaging of LDs, in contrast to the behavior of cationic styrylpyridinium (TNC), which primarily localizes within the mitochondria. Notably, pyridine N-oxide offers several benefits: an increased dipole moment facilitating charge separation between donors and acceptors, substantial HOMO and LUMO stabilization, improved water solubility, favorable redox properties, and bathochromic-shifted absorption/emission spectra, showing promise as a fluorescent tool for probing the cellular-biological realm.

Improved lipophilic probe for visualizing lipid droplets in erastin-induced ferroptosis.

Studying the viscosity of lipid droplets (LDs) provides insights into various diseases associated with LD viscosity. Ferroptosis is one such process in which LD viscosity increases due to the abnormal accumulation of lipid ROS (reactive oxygen species) caused by peroxidation. For investigating the LD imaging and ferroptosis, we developed two molecules (NNS and DNS) that show significant Stokes shifts (182-232 nm) and utilized them for sub-cellular imaging. Excellent localization is noted with the lipid droplets. Subsequently, DNS was used to monitor the variations in the LD viscosity during erastin-induced ferroptosis followed by ferroptosis inhibition. Additionally, we explored variations in the LD quantity, size, and accumulation when subjected to oleic acid stimulation. Extensive DFT and TDDFT investigations have been employed to understand the effect of NO2 substitution on the linear and branched molecular derivatives. Our results with the improved lipophilic fluorophore, exhibiting excellent colocalization with LDs, offer valuable insights into sensing erastin-induced ferroptosis and have the potential for real-time diagnostic applications.

N-Functionalized fluorophores: detecting urinary albumin and imaging lipid droplets.

A series of novel N-sulfonyl pyridinium fluorophores were designed, synthesized, and explored in terms of their ability to bind with serum albumins. Upon binding the fluorophores with BSA, noticeable emission wavelength or intensity changes accompanied by color changes were observed. Competitive binding studies revealed that the fluorophore selectively binds to the warfarin site, but the binding affinity also depends on the nature of the scaffold. Additionally, the fluorophores were employed to detect spiked serum albumin in artificial urine. Cellular imaging experiments indicated that the fluorophores accumulate within lipid droplets (LDs), suggesting their potential as promising biomarkers for lipid droplets. Furthermore, the fluorescence intensity, number, and size of LDs increased upon serum starvation.

Styryl-NIR Mitochondrial Probes with Rapid HOCl Detection in Live-Cells.

Hypochlorous acid (HOCl) is critical for maintaining immune system balance, but it can harm mitochondria by hindering enzyme activity, leading to decreased ATP and increased cell death. In this study, we have designed a fluorophore with a pyridinium scaffold for selective staining of the mitochondria and to detect hypochlorite. The fluorophore exhibits strong solvatochromic emission due to intramolecular charge transfer and excellent sub-cellular localization in the mitochondria. Additionally, it shows a rapid response to HOCl with high selectivity among different reactive oxygen/nitrogen compounds with a detection limit of 2.31 µM. Moreover, it is also utilized for the exogenous and endogenous detection of HOCl in live cells, which may help study the role of hypochlorite in organelles at the cellular level. DFT and TDDFT calculations have been carried out to understand the relationship between the structure and properties of the cationic probes with respect to the α-cyano substitution and extension of π-conjugation. The selective detection of HOCl by C4 over other cationic probes has also been well-demonstrated, showing how the binding of HOCl affects the electronic properties of C4 through the analysis of non-bonding orbitals (NBO) population, electrostatic potential surface (ESP), and density of states (DOS) projected DOS investigations.

Imaging of mitochondria/lysosomes in live cells and C. elegans.

Two rhodamine-phenothiazine conjugates, RP1 and RP2, were synthesized, and their photophysical properties, subcellular localization, and photocytotoxicity were investigated. We observed robust localization of RP1 in mitochondria and dual localization in mitochondria and lysosomes with RP2 in live cells. Live cell imaging with these probes allowed us to track the dynamics of mitochondria and lysosomes during ROS-induced mitochondrial damage and the subsequent lysosomal digestion of the damaged mitochondria. The fluorophores also demonstrated preferential accumulation in cancer cells compared to normal cells and had strong photo-cytotoxicity. However, no cytotoxicity was observed in the dark. The mitochondrial staining and light-induced ROS production were not limited to mammalian cell lines, but were also observed in the animal model C. elegans. The study demonstrated the potential applications of these probes in visualizing the mitochondria-lysosome cross-talk after ROS production and for photodynamic therapy.

Imaging of lipid droplets using coumarin fluorophores in live cells and C. elegans.

Fluorescent probes offer incredibly effective tools for visualizing the dynamic morphology of lipid droplets (LDs) and investigating their physiological interactions. In this work, we have utilized solvatochromic coumarin probes bearing nitrile and ester substituents for live-cell imaging. The fluorescence probes are characterized by a donor (diethylamino) and acceptor (nitrile and/or ester) substituents and a rotatable double bond. The designed architecture allows investigation of environmental sensitivity apart from providing excellent ability to target sub-cellular organelles. The synthesized fluorophores showed low cytotoxicity and excellent localization within the lipid droplets. Further, the fluorophores were also utilized to study viscosity changes within the LDs induced by Nystatin. More importantly, we also demonstrate imaging of LDs in multi-cellular animal models such as C. elegans.

Fluorescent sterol probes for intracellular transport, imaging, and therapeutics.

Sterols play a significant role in many physiological processes affecting membrane organization, transport, permeability, and signal transduction. The development of fluorescent sterol analogs that have immediate functional relevance to the natural biomolecules is one approach to understanding the sterol-driven physiological processes. Visualizing cellular compartments with tailor-made fluorescent molecules through specific labeling methods enables organelle targeting and reveals dynamic information. In this review, we focus on the recent literature published between 2020 and 2022, with particular emphasis on extrinsic fluorophores and their investigations of sterol-driven biological processes involving sterol transport, biomolecular interactions, and biological imaging.

α-Cyanostilbene: a multifunctional spectral engineering motif.

Aggregation-induced emission (AIE) is a unique photophysical phenomenon of organic chromophores, exhibiting a significant emission enhancement in the condensed phase (aggregate/solid/film) than in the solution phase. This remarkable feature offers excellent strategies to obtain molecular materials possessing unique spectral signatures such as high fluorescence intensity, excellent quantum yield, large Stokes shift, and exquisite optoelectronic properties. Unlike a great library of articles with propeller-shaped tetraphenylethene molecular frameworks, reviews based on the mechanistic understandings of α-cyanostilbenes are relatively rare. Considering this, herein, we highlight the structure-property relationship of α-cyanostilbene-based AIE frameworks for tuning the aggregation through molecular displacement with reference to transition dipoles based on the following parameters: (i) positional substitution and orientation of the α-cyano unit, (ii) π-conjugation length (da or db), (iii) molecular size (DAr) of the peripheral substitutions with respect to the α-cyano unit, and (iv) branching effect. In addition, we explain the utility of their unique AIE characteristics for various optoelectronic applications, including self-assembled nanostructures, chemical sensing, organogelation, white light emission, molecular switches, multiphoton absorption, liquid crystals, anion receptors, and biological probes. It is anticipated that organic materials with a cyanostilbene framework will continue to garner attention in the interdisciplinary fields of biology, chemistry, and materials science for diverse applications.

Lutidine derivatives for live-cell imaging of the mitochondria and endoplasmic reticulum.

The mitochondria and endoplasmic reticulum (ER) are highly dynamic subcellular structures essential for several biological functions. The development of non-toxic, wash-free fluorophores to visualize these structures inside cells aid in understanding their localization and dynamics in diverse cellular processes. In this paper, we report the synthesis and characterization of lutidine-based cationic fluorophores bearing push-pull substituents exhibiting emission in green and red wavelength regions and their subcellular localization in living cells. The confocal imaging of the molecules in a cellular domain reveals the robust localization of three molecules (2, 4 and 5) in the mitochondria and two molecules with polyfluorophenyl substituents (6 and 7) in the ER. At the same time, the other two molecules (1 and 3) showed non-specific imaging. These molecules can also be used to sense the altered viscosity of the stressed ER and investigate its dynamics.

Fluorescent probes for targeting endoplasmic reticulum: design strategies and their applications.

Advances in developing organic fluorescent probes and fluorescence imaging techniques have enhanced our understanding of cell biology. The endoplasmic reticulum (ER) is a dynamic structure that plays a crucial role in protein synthesis, post-translational modifications, and lipid metabolism. The malfunction of ER contributes to several physiological and pathological conditions. Therefore, the investigations on the imaging and role of ER have attracted a lot of attention. Due to their simplicity, synthetic tunability, photostability, high quantum yields, easier cellular uptake, and lower cytotoxicity, organic fluorophores offer invaluable tools for the precision targeting of various cellular organelles and probe ER dynamics. The precision staining is made possible by incorporating specific functional groups having preferential and local organelle biomolecular interactions. For instance, functional moieties such as methyl sulfonamide, sulfonylurea, and pentafluorophenyl assist in ER targeting and thus have become essential tools to probe a deeper understanding of their dynamics. Furthermore, dual-function fluorescent probes that simultaneously image ER and detect specific physiological parameters or biological analytes were achieved by introducing special recognition or chemically reactive sites. This article attempts to comprehensively capture various design strategies currently employed by researchers utilizing small organic molecules to target the ER and detect specific analytes.

Stress-responsive rhodamine bioconjugates for membrane-potential-independent mitochondrial live-cell imaging and tracking.

The 'powerhouses' of cell, mitochondria have seen an upsurge of interest in investigations pertaining to the imaging and mapping of physiological processes. By utilizing sterol-modified rhodamine, we have performed the live-cell imaging of mitochondria without dependence on a membrane potential. The sterol probes are highly biocompatible, and they can track the mitochondrial live-cell dynamics in a background-free manner with improved brightness and impressive contrast. This is the first attempt to study the stress response using a direct fluorescence readout with bio-conjugates of rhodamine inside mitochondria. The results pave the way for developing different sterol markers for understanding cellular responses and function.

Probing Variations of Reduction Activity at the Plasma Membrane Using a Targeted Ratiometric FRET Probe.

Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.

Live-cell imaging of the nucleolus and mapping mitochondrial viscosity with a dual function fluorescent probe.

Visualization of sub-cellular organelles allows the determination of various cellular processes and the underlying mechanisms. Herein, we report a fluorescent probe, bearing push-pull substituents emitting at 600 nm and its application in cellular imaging. The probe shows dual imaging of mitochondria and nucleoli and maps mitochondrial viscosity in live cells under various physiological variations and show minimum cytotoxicity. Nucleolar staining is confirmed by RNAase digestion.