Inter-organellar interactions play indispensable roles in regulating cellular homeostasis, necessitating advanced methodologies for their simultaneous and discriminative visualization. Fluorescent probes, prized for their sensitivity and spatiotemporal resolution, are pivotal tools for elucidating organelle dynamics in live-cell studies. However, current technologies remain limited in achieving robust dual-color imaging of multiple organelles with minimal crosstalk. To address this gap, we developed a Förster resonance energy transfer (FRET)-based ratiometric probe leveraging the pH-responsive spiro-pyran motif, which undergoes reversible ring-opening/closing transitions. This probe enables concurrent dual-color visualization of lipid droplets (LDs) and the endoplasmic reticulum (ER) in HeLa cells under single-excitation conditions, achieving high Pearson's correlation coefficients and minimal spectral overlap. Our work advances the design of multifunctional probes for decoding inter-organelle communication in live systems.

The design of small-molecule fluorescent probes for labelling the endoplasmic reticulum (ER) revolves around a well-established albeit limited group of structural architectures. Here, we synthesized new fluorescent lophines in one step in high-temperature water (HTW) and explored their application as dyes for selective bioimaging of the ER.

Changes in viscosity can significantly influence the functionality of the endoplasmic reticulum (ER), and viscosity-responsive fluorescent probes can provide valuable feedback on its physiological state. In this study, three multirotor-based fluorescent probes (1-3) were designed and synthesized. Probes 1 and 2 exhibited remarkable viscosity sensitivity and specific targeting ability towards the ER. Probe 1 was utilized for in situ dynamic visualization of ER viscosity changes during inflammatory responses, drug treatments, and reticulophagy.

Multi-organelle imaging allows the visualization of multiple organelles within a single cell, allowing monitoring of the cellular processes in real-time using various fluorescent probes that target specific organelles. However, the limited availability of fluorophores and potential spectral overlap present challenges, and many optimized designs are still in nascency. In this work, we synthesized various sulfonamide-based organic fluorophores that emit in the blue, green, and red regions to target different sub-cellular organelles. By utilizing binary mixtures, we successfully demonstrated multiple imaging of the sub-cellular organelles, such as the endoplasmic reticulum, plasma membrane, and mitochondria in HeLa cells, and dual imaging of the endoplasmic reticulum and mitochondria in A549 lung carcinoma cells with the help of blue and red-emitting fluorophores without any spectral spillover. Additionally, these photostable probes allowed precise cell staining and differentiation, structural features, and live cell dynamics. This approach of utilizing fluorescent mixtures can gain traction for various cellular studies and investigations.

Endoplasmic reticulum (ER) stress and autophagy (ER-phagy) occurring in nerve cells are crucial physiological processes closely associated with Alzheimer's disease (AD). Visualizing the two processes is paramount to advance our understanding of AD pathologies. Among the biomarkers identified, peroxynitrite (ONOO-) emerges as a key molecule in the initiation and aggravation of ER stress and ER-phagy, highlighting its significance in the underlying mechanisms of the two processes. In this work, we designed and synthesized an innovative ONOO--responsive AIEgen-based fluorescent probe (DHQM) with the ability to monitor ER stress and ER-phagy in AD model cells. DHQM demonstrated excellent aggregation-induced emission (AIE) properties, endowing it with outstanding ability for washing-free intracellular imaging. Meanwhile, it exhibited high sensitivity, remarkable selectivity to ONOO-, and exceptional ER-targeting ability. The probe was successfully applied for fluorescence imaging of ER ONOO- fluctuations to assess the ER stress status in aluminum-induced AD model cells. Our findings revealed that aluminum-induced ferroptosis, a regulated cell death process, was pivotal in the excessive ONOO- production, which in turn activated and exacerbated ER stress. Furthermore, the aluminum-stimulated ER-phagy was observed utilizing DHQM, which might be crucial in inhibiting ferroptosis and mitigating aberrant ER stress. Overall, this study not only offers valuable insights into the pathological mechanisms of AD at the ER level but also opens new potential therapeutic avenues targeting these pathways.

We report a bithiophene-based fluorescence probe BDT (2,2'-(((1 E, 1'E)-[2,2'-bithiophene]-5,5'-diylbis(methaneylylidene))bis(azaneylylidene))bis(4-(tert-butyl)phenol)) for recognizing ClO-. BDT selectively responded to ClO-, leading to a blue fluorescence enhancement in a mixture of DMF/HEPES buffer (9:1, v/v). Importantly, BDT showed an ultrafast response (within 1 s) to ClO- among the fluorescent turn-on chemosensors based on bithiophene. BDT recognized ClO- through cleavage reaction with a low detection limit of 2.16 µM, and it had the ability to sense ClO- across a pH range of 3-11. The recognition mechanism for ClO- was investigated by 1H nuclear magnetic resonance (NMR) titration, electrospray ionization mass spectrometry (ESI-MS), and density functional theory (DFT) calculations. In addition, BDT could be used to detect ClO- using test strips as a convenient tool, allowing real-time monitoring rapidly. Practically, BDT exhibited reliable recoveries for quantifying ClO- using a smartphone application with a spike-and-recovery method in real water samples such as drinking, tap, mineral, and river water.

The significance of H2O2 as a marker of reactive oxygen species (ROS) and oxidative stress in living organisms has spurred growing interest in its roles in inflammation and disease progression. In this report, a ratiometric time-gated luminescence (RTGL) probe is proposed based on mixed lanthanide complexes, ER-BATTA-Tb3+/Eu3+, for imaging the H2O2 generation both in vitro and in vivo. Upon exposure to H2O2, the probe undergoes cleavage of the benzyl boric acid group, releasing hydroxyl (─OH) groups, which significantly reduces the emission of the Eu3+ complex while slightly increasing the emission of the Tb3+ complex. This response allows the I540/I610 ratio to be used as an indicator for monitoring the H2O2 level changes. The probes are capable of selectively accumulating in the endoplasmic reticulum (ER), allowing effective imaging of H2O2 in the ER of living cells and liver-injured mice under oxidative stress. Moreover, by integrating ER-BATTA-Tb3+/Eu3+ into (polyethylene glycol) PEG hydrogels, the H2O2-responsive smart sensor films, PEG-H2O2-Sensor films, are created, which enable the real-time monitoring of H2O2 levels in various wounds using a smartphone imaging platform and R/G channel evaluation. The sensor films are also innovatively applied for the in situ monitoring of H2O2 in brains of epileptic rats, facilitating the precise assessment of brain damage. This study provides a valuable tool for the quantitative detection of H2O2 in vitro and in vivo, as well as for the clinical monitoring and treatment of H2O2-related diseases in multiple scenarios.

The crucial cellular activities for maintaining normal cell functions heavily rely on the polarity of the endoplasmic reticulum (ER). Understanding how the polarity shifts, particularly in the context of ER autophagy (ER-phagy), holds significant promise for advancing knowledge of disorders associated with ER stress. Herein, a polarity-sensitive fluorescent probe CDI was easily synthesized from the condensation reaction of coumarin and dicyanoisophorone. CDI was composed of coumarin as the electron-donating moiety (D), ethylene and phenyl ring as the π-conjugation bridge, and malononitrile as the electron-accepting moiety (A), forming a typical D-π-A molecular configuration that recognition in the near-infrared (NIR) region. The findings suggested that as the polarity increased, the fluorescence intensity of CDI decreased, and it was accompanied by a redshift of emission wavelength at the excitation wavelength of 524 nm, shifting from 641 nm to 721 nm. Significantly, CDI exhibited a notable ability to effectively target ER and enabled real-time monitoring of ER-phagy induced by starvation or drugs. Most importantly, alterations in polarity can be discerned through in vivo imaging in mice model of rheumatoid arthritis (RA). CDI has been proven effective in evaluating the therapeutic efficacy of drugs for RA. ER fluorescent probe CDI can be optically activated in lysosomes, providing a sensitive tool for studying ER-phagy in biology and diseases.

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.

Beta-hydroxybutyrate (β-HB) serve as a valuable diagnostic biomarker for Diabetic Ketoacidosis (DKA). Here, a new Schiff base fluorescent probe T was designed and synthesized to detect β-HB level in aqueous solution in vitro. The probe T can detect β-HB sensitively and selectively in DMF solution (5.0 × 10-5 M) among other interfering species (cations, anions, amino acids, biomarkers). The detection limit of probe T for β-HB was calculated to be 0.154 μM. These results demonstrate that the probe T may provide a convenient method for rapid detection of β-HB to diagnose diabetic ketoacidosis.

It is evident that site-specific systemic drug delivery can reduce side effects, systemic toxicity, and minimal dosage requirements predominantly by delivering drugs to particular pathological sites, cells, and even subcellular structures. The endoplasmic reticulum (ER) and associated cell organelles play a vital role in several essential cellular functions and activities, such as the synthesis of lipids, steroids, membrane-associated proteins along with intracellular transport, signaling of Ca2+, and specific response to stress. Therefore, the dysfunction of ER is correlated with numerous diseases where cancer, neurodegenerative disorders, diabetes mellitus, hepatic disorder, etc., are very common. To achieve satisfactory therapeutic results in certain diseases, it is essential to engineer delivery systems that can effectively enter the cells and target ER. Nanoparticles are highly biocompatible, contain a variety of cargos or payloads, and can be modified in a pliable manner to achieve therapeutic effectiveness at the subcellular level when delivered to specific organelles. Passive targeting drug delivery vehicles, or active targeting drug delivery systems, reduce the nonselective accumulation of drugs while reducing side effects by modifying them with small molecular compounds, antibodies, polypeptides, or isolated bio-membranes. The targeting of ER and closely associated organelles in cells using nanoparticles, however, is still unsymmetrically understood. Therefore, here we summarized the pathophysiological prospect of ER stress, involvement of ER and mitochondrial response, disease related to ER dysfunctions, essential therapeutics, and nanoenabled modulation of their delivery to optimize therapy.

RNA structures, including those formed from coding and noncoding RNAs, alternative to protein-based drug targets, could be a promising target of small molecules for drug discovery against various human diseases, particularly in anticancer, antibacterial and antivirus development. The normal cellular activity of cells is critically dependent on the function of various RNA molecules generated from DNA transcription. Moreover, many studies support that mRNA-targeting small molecules may regulate the synthesis of disease-related proteins via the non-covalent mRNA-ligand interactions that do not involve gene modification. RNA-ligand interaction is thus an attractive approach to address the challenge of "undruggable" proteins in drug discovery because the intracellular activity of these proteins is hard to be suppressed with small molecule ligands. We selectively surveyed a specific area of RNA structure-selective small molecule ligands in fluorescence live cell imaging and drug discovery because the area was currently underexplored. This state-of-the-art review thus mainly focuses on the research published within the past three years and aims to provide the most recent information on this research area; hopefully, it could be complementary to the previously reported reviews and give new insights into the future development on RNA-specific small molecule ligands for live cell imaging and drug discovery.

The alleviation of drug-induced liver injury has been a long-term public health concern. Growing evidence suggests that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of drug-induced hepatotoxicity. Therefore, the inhibition of ER stress has gradually become one of the important pathways to alleviate drug-induced liver injury. In this work, we developed an ER-targeted photoreleaser, ERC, for controllable carbon monoxide (CO) release with a near-infrared light trigger. By employing peroxynitrite (ONOO^-) as an imaging biomarker of hepatotoxicity, the remediating effect of CO was mapped upon drug acetaminophen (APAP) challenge. The direct and visual evidence of suppressing oxidative and nitrosative stress by CO was obtained both in living cells and in mice. Additionally, the ER stress inhibiting the effect of CO was verified during drug-induced hepatotoxicity. This work demonstrated that CO may be employed as a potent potential antidote for APAP-related oxidative and nitrative stress remediation.

Hypochlorite (ClO^-) plays an important role in the human immune defense system, but high concentrations of ClO^- in the endoplasmic reticulum (ER) damage cellular proteins, causing ER stress, cell death, and various diseases. Herein, we developed a simple hydrazone probe (1) featuring aggregation-induced ratiometric emission, which would quickly (within 20 s) and sensitively (detection limit of 15.4 μM) respond to ClO^- in an almost pure aqueous solution via a fluorescent ratiometric output. Furthermore, the probe was employed to track the level of ClO^- in the ER of HeLa cells and zebrafish.

In the last decades, new evidence on brain structure and function has been acquired by morphological investigations based on synergic interactions between biochemical anatomy approaches, new techniques in microscopy and brain imaging, and quantitative analysis of the obtained images. This effort produced an expanded view on brain architecture, illustrating the central nervous system as a huge network of cells and regions in which intercellular communication processes, involving not only neurons but also other cell populations, virtually determine all aspects of the integrative function performed by the system. The main features of these processes are described. They include the two basic modes of intercellular communication identified (i.e., wiring and volume transmission) and mechanisms modulating the intercellular signaling, such as cotransmission and allosteric receptor-receptor interactions. These features may also open new possibilities for the development of novel pharmacological approaches to address central nervous system diseases. This aspect, with a potential major impact on molecular medicine, will be also briefly discussed.