Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis.

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

3D Imaging of Lipid Droplet-Nuclear Membrane Contact Sites and Cirrhotic Lipid Droplet Overexpression.

To coordinate cellular physiology, cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Lipid droplets (LDs) and nuclear membrane (NM) contact sites are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites. However, there is still a lack of understanding of the specific morphology of the contact sites. Here, we combine advanced three-dimensional (3D) imaging with a high-brightness fluorescent probe specifically targeting LDs to map the structural landscape of LD-NM contact sites. The probe exhibits exceptional photophysical properties, making it highly suitable for visualizing the changes occurring in LDs during the apoptosis process. In addition, we utilize the advantages of the probe to accurately monitor the overexpression of abnormal LDs in cirrhosis by 3D imaging for the first time. The outcomes of this investigation highlight that the probe has potential as a robust imaging tool to investigate intricate biological functions of LDs and their implications in related diseases.

Activatable Fluorescent Probe for Studying Drug-Induced Senescence In Vitro and In Vivo.

Aging represents a significant risk factor for compromised tissue function and the development of chronic diseases in the human body. This process is intricately linked to oxidative stress, with HClO serving as a vital reactive oxygen species (ROS) within biological systems due to its strong oxidative properties. Hence, conducting a thorough examination of HClO in the context of aging is crucial for advancing the field of aging biology. In this work, we successfully developed a fluorescent probe, OPD, tailored specifically for detecting HClO in senescent cells and in vivo. Impressively, OPD exhibited a robust reaction with HClO, showcasing outstanding selectivity, sensitivity, and photostability. Notably, OPD effectively identified HClO in senescent cells for the first time, confirming that DOX- and ROS-induced senescent cells exhibited higher HClO levels compared to uninduced normal cells. Additionally, in vivo imaging of zebrafish demonstrated that d-galactose- and ROS-stimulated senescent zebrafish displayed elevated HClO levels compared to normal zebrafish. Furthermore, when applied to mouse tissues and organs, OPD revealed increased fluorescence in the organs of senescent mice compared to their nonsenescent counterparts. Our findings also illustrated the probe's potential for detecting changes in HClO content pre- and post-aging in living mice. Overall, this probe holds immense promise as a valuable tool for in vivo detection of HClO and for studying aging biology in live organisms.

Mitochondria-Specific Fluorescent Probe for Revealing the Interaction between Mitochondria and Lysosomes during Apoptosis.

The mitochondria, as one of the essential organelles in cells, are closely associated with numerous biological processes. Therefore, the realization of clear and real-time imaging for tracking mitochondria is of profound significance. Here, we present a mitochondria-targeting fluorescent probe, N(CH2)3-PD-NEt, for the real-time fluorescence imaging of mitochondria in living cells. Using the probe, the fluorescence changes of mitochondria stimulated by different drugs were successfully observed by fluorescence imaging. In addition, the dynamic processes of mitochondria and lysosomes during apoptosis were also explored. Importantly, we observed several novel dynamic interaction patterns between mitochondria and lysosomes. Among them, the most prominent pattern involved the noncontact movements of two lysosomes, that is, one lysosome gradually approached the other lysosome over time, eventually coming into contact and merging with it while gradually combining with mitochondria to form new mitochondria. Notably, the protrusions of the mitochondria became increasingly evident during this process. Meanwhile, we successfully observed the dynamic changes of mitochondria with SIM super-resolution imaging. The study provides promising help for the in-depth study of the dynamic processes of mitochondrial physiology and pathology and the study of the interactions between organelles.

Cascaded and Localized Assembly of DNA Nanospheres for Efficient mRNA Imaging.

The pursuit of advanced mRNA detection methods has been driven by the need for sensitive, accurate approaches that are particularly suited for live-cell analysis. Herein, we proposed a cascaded and localized assembly (CLA) system, integrating branched catalytic hairpin assembly (bCHA) with a localized hybridization chain reaction (LHCR) for enhanced mRNA imaging. The CLA system employed a dual-nanosphere (NS) platform, NSABC and NS12, and the interaction between the target and NSABC initiated the bCHA process and activated a split trigger. The newly generated trigger served as the initiator for the LHCR on NS12, leading to amplified fluorescent signals. Notably, this work introduced the first integration of a splitting strategy in a bCHA-HCR cascaded system, reducing false-positive signals and enhancing specific detection. The dual-NS platform further minimized background noise and improved the reaction kinetics through spatial confinement. As a result, the system achieved a detection limit of 1.23 pM. With these advantages, the CLA system demonstrated successful application in both living cells and clinical tissues, underscoring its potential in biomolecular research and clinical diagnostics.

Construction of a Label-Detection Integrated Visual Probe to Reveal the Double-Edged Sword Principle of Ferroptosis in Liver Injury.

Ferroptosis has been confirmed as a potential mediator and an indicator of the severity of liver injury. Despite the fruitful results, there are still two deficiencies in the research on the association between ferroptosis and liver injury. First, iron ions are usually selected as the target bioanalyte, but its detection based on a fluorescent probe is interfered with by specific chemical reaction mechanisms, leading to low sensitivity and poor physiological stability. Second, more efforts were focused on the harmful effects of ferroptosis on liver injury and less involved in the therapeutic value of ferroptosis for liver injury. Hence, in this work, we proposed a new nonreactive analyte (mitochondrial viscosity) as an analysis marker, which can circumvent the challenges caused by specific reaction mechanisms of iron ions. Meanwhile, we constructed a novel label-detection integrated visual probe (VPF) to explore the feasibility of ferroptosis in the treatment of liver injury. As expected, we not only successfully traced the dynamic changes in mitochondrial viscosity but also visualized the changes in cell morphology during induced and inhibited ferroptosis. Conspicuously, this work revealed that liver injury can be alleviated by regulating ferroptosis, confirming the therapeutic value of ferroptosis in liver injury. In addition, a complex biological communication network between ferroptosis and liver injury was constructed by western blotting, providing an important theoretical mechanism for revealing their double-edged sword relationship. This study not only provides a new strategy for studying the complex relationship between ferroptosis and liver injury but also facilitates the future treatment of liver injury.

An efficient lipid droplet-targeted fluorescent probe for detection of intracellular viscosity.

Lipid droplet, an intracellular lipid reservoir, is vital for energy metabolism and signal transmission in cells. The viscosity directly affects the metabolism of lipid droplets, and the abnormal viscosity is associated with the occurrence and development of various diseases. Therefore, it is indispensable to develop techniques that can detect viscosity changes in intracellular lipid droplets. Based on twisted intramolecular charge transfer (TICT) mechanism, a novel small-molecule lipid droplet-targeted viscosity fluorescence probe PPF-1 was designed. The probe was easy to synthesize, it had a large Stokes shift, stable optical properties, and low bio-toxicity. Compared to being in methanol solution, the fluorescence intensity of PPF-1 in glycerol solution was increased 26.7-fold, and PPF-1 showed excellent ability to target lipid droplets. Thus, the probe PPF-1 could provide an effective means of detecting viscosity changes of lipid droplets and was of great value for physiological diagnosis of related diseases, pathological analysis, and medical research.

Accelerated diagnosis: a crosslinking catalytic hairpin assembly system for rapid and sensitive SARS-CoV-2 RNA detection.

The COVID-19 pandemic has underscored the urgent need for rapid and reliable strategies for early detection of SARS-CoV-2. In this study, we propose a DNA nanosphere-based crosslinking catalytic hairpin assembly (CCHA) system for the rapid and sensitive SARS-CoV-2 RNA detection. The CCHA system employs two DNA nanospheres functionalized with catalytic hairpin assembly (CHA) hairpins. The presence of target SARS-CoV-2 RNA initiated the crosslinking of DNA nanospheres via CHA process, leading to the amplification of fluorescence signals. As a result, the speed of SARS-CoV-2 diagnosis was enhanced by significantly increasing the local concentration of the reagents in a crosslinked DNA product, leading to a detection limit of 363 fM within 5 min. The robustness of this system has been validated in complex environments, such as fetal bovine serum and saliva. Hence, the proposed CCHA system offers an efficient and simple approach for rapid detection of SARS-CoV-2 RNA, holding substantial promise for enhancing COVID-19 diagnosis.

Fluorescent probes for ferroptosis bioimaging: advances, challenges, and prospects.

Ferroptosis is a form of regulatory cell death distinct from caspase-dependent apoptosis and plays an important role in life entities. Since ferroptosis involves a variety of complex regulatory factors, the levels of certain biological species and microenvironments would change during this process. Thus, the investigation of the level fluctuation of key target analytes during ferroptosis is of great significance for disease treatment and drug design. Toward this aim, multiple organic fluorescent probes with simple preparation and non-destructive detection have been developed, and research over the past decade has uncovered a vast array of homeostasis and other physiological characteristics of ferroptosis. However, this significant and cutting-edge topic has not yet been reviewed. In this work, we aim to highlight the latest breakthrough results of fluorescent probes for monitoring various bio-related molecules and microenvironments during ferroptosis at the cellular, tissue and in vivo levels. Accordingly, this tutorial review has been organized according to the target molecules identified by the probes including ionic species, reactive sulfur species, reactive oxygen species, biomacromolecules, microenvironment, and others. In addition to providing new insights into the findings of each fluorescent probe in ferroptosis studies, we also discuss the defects and limitations of the probes developed, and highlight the potential challenges and further prospects in this domain. We anticipate that this review will convey profound implications for designing powerful fluorescent probes to decrypt changes in key molecules and microenvironments during ferroptosis.

Development of a Fluorescent Probe with High Selectivity based on Thiol-ene Click Nucleophilic Cascade Reactions for Delving into the Action Mechanism of Serotonin in Depression.

The intrinsic correlation between depression and serotonin (5-HT) is a highly debated topic, with significant implications for the diagnosis, treatment, and advancement of drugs targeting neurological disorders. To address this important question, it is of utmost priority to understand the action mechanism of serotonin in depression through fluorescence imaging studies. However, the development of efficient molecular probes for serotonin is hindered by the lack of responsive sites with high selectivity for serotonin at the present time. Herein, we developed the first highly selective serotonin responsive site, 3-mercaptopropionate, utilizing thiol-ene click cascade nucleophilic reactions. The novel responsive site was then employed to construct the powerful molecular probe SJ-5-HT for imaging the serotonin level changes in the depression cells and brain tissues. Importantly, the imaging studies reveal that the level of serotonin in patients with depression may not be the primary factor, while the ability of neurons in patients with depression to release serotonin appears to be more critical. Additionally, this serotonin release capability correlates strongly with the levels of mTOR (intracellular mammalian target of rapamycin). These discoveries could offer valuable insights into the molecular mechanisms underpinning depression and furnish mTOR as a novel direction for the advancement of antidepressant therapies.

A Class of Activatable NIR-II Photoacoustic Dyes for High-Contrast Bioimaging.

Photoacoustic (PA) imaging is emerging as one of the important non-invasive imaging techniques in biomedical research. Small molecule- second near-infrared window (NIR-II) PA dyes combined with imaging data can provide comprehensive and in-depth in vivo physiological and pathological information. However, the NIR-II PA dyes usually exhibit "always-on" properties due to the lack of a readily optically tunable group, which hinders the further applications in vivo. Herein, a novel class of dyes GX have been designed and synthesized as an activatable NIR-II PA platform, in which the absorption/emission wavelength of GX-5 extends up to 1082/1360 nm. Importantly, the GX dyes have a strong tissue penetration depth and high-resolution for the mouse vasculature structures in NIR-II PA 3D imaging and high signal-to-noise ratio in NIR-II fluorescence (FL) imaging. Furthermore, to demonstrate the applicability of GX dyes, the first NIR-II PA probe GX-5-CO activated by carbon monoxide (CO) was engineered and employed to reveal the enhancement of the CO levels in the hypertensive mice by high-contrast NIR-II PA and FL imaging. We expect that many derivatives of GX dyes will be developed to afford versatile NIR-II PA platforms for designing a wide variety activatable NIR-II PA probes as biomedical tools.

Investigating the Therapeutic Effects of Ferroptosis on Myocardial Ischemia-Reperfusion Injury Using a Dual-Locking Mitochondrial Targeting Strategy.

Research on ferroptosis in myocardial ischemia/reperfusion injury (MIRI) using mitochondrial viscosity as a nexus holds great promise for MIRI therapy. However, high-precision visualisation of mitochondrial viscosity remains a formidable task owing to the debilitating electrostatic interactions caused by damaged mitochondrial membrane potential. Herein, we propose a dual-locking mitochondria-targeting strategy that incorporates electrostatic forces and probe-protein molecular docking. Even in damaged mitochondria, stable and precise visualisation of mitochondrial viscosity in triggered and medicated MIRI was achieved owing to the sustained driving forces (e.g., pi-cation, pi-alkyl interactions, etc.) between the developed probe, CBS, and the mitochondrial membrane protein. Moreover, complemented by a western blot, we confirmed that ferrostatin-1 exerts its therapeutic effect on MIRI by improving the system xc-/GSH/GPX4 antioxidant system, confirming the therapeutic value of ferroptosis in MIRI. This study presents a novel strategy for developing robust mitochondrial probes, thereby advancing MIRI treatment.

Three-Dimensional CHA-HCR System Using DNA Nanospheres for Sensitive and Rapid Imaging of miRNA in Live Cells and Tissues.

Isothermal, enzyme-free amplification techniques, such as the hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), have gained increasing attention for miRNA analysis. However, current methodological challenges, including slow kinetics, low amplification efficiency, difficulties in efficient cellular internalization of DNA probes, and concerns regarding the intracellular stability of nucleic acids, need to be addressed. To this end, we propose a novel strategy for sensitive miRNA detection based on a three-dimensional (3D) CHA-HCR system. This system comprises two DNA nanospheres, named DS-13 and DS-24, which are functionalized with CHA and HCR hairpins. Target miR-21 initiates CHA between the two nanospheres, thereby activating downstream HCR and bringing cyanine 3 (Cy3) and cyanine 5 (Cy5) into proximity. The 3D CHA-HCR process leads to the formation of large DNA aggregates and the generation of fluorescence resonance energy transfer signals. In this strategy, the employment of a cascaded reaction and spatial confinement effect improve sensitivity and kinetics, while the use of DNA nanocarriers facilitates cellular delivery and protects nucleic acid probes. The experimental results in vitro, in living cells, and in clinical tissue samples demonstrated the desirable sensing performance. Collectively, this approach holds promise as a valuable tool for cancer diagnosis and biomedical research.

Hierarchical MOF@AuNP/Hairpin Nanotheranostic for Enhanced Photodynamic Therapy via O2 Self-Supply and Cancer-Related MicroRNA Imaging In Vivo.

Developing a nanotheranostic with a high sensing performance and efficient therapy was significant in cancer diagnosis and treatment. Herein, a Au nanoparticle and hairpin-loaded photosensitive metal-organic framework (PMOF@AuNP/hairpin) nanotheranostic was constructed by growing AuNPs on PMOF in situ and then attaching hairpins. On the one hand, the PMOF@AuNP/hairpin nanotheranostic could effectively transfer O2 into ROS, facilitating efficient PDT. Additionally, the nanotheranostic possessed catalase-like activity, which could effectively catalyze H2O2 to generate O2, thus achieving O2-evolving PDT and significantly enhancing the antitumor effect of PDT in vivo. On the other hand, the nanotheranostic showed a high loading efficiency of hairpins and achieved the sensitive and selective detection of miR-21 both in living cells and in vivo. Moreover, the nanotheranostic could dynamically monitor the miR-21 level. Due to the excellent imaging performance, the nanotheranostic could recognize cancer cells and might provide important information on cancer progression for PDT. The developed PMOF@AuNP/hairpin nanotheranostic provided a useful tool for tumor diagnosis and antitumor therapy.

Strategy for Fluorescence/Photoacoustic Signal Maximization Using Dual-Wavelength-Independent Excitation.

Dual-mode imaging of fluorescence-photoacoustics has emerged as a promising technique for biomedical applications. However, conventional dual-mode imaging is based on single-wavelength excitation, which often results in opposing fluorescence and photoacoustic signals due to competing photophysical processes in one agent, rendering the maximization of both signals infeasible. To meet this challenge, we herein propose a new strategy by using the dual-excitation approach, where one excitation wavelength generates a fluorescence signal and the other produces a photoacoustic signal, thus achieving simultaneous maximization of both signals in one fluorescence-photoacoustic molecule. Based on this strategy, three dye molecules were employed for comparison, and it was surprising to find that QHD dye with two types of excitation wavelengths could generate fluorescence and photoacoustic signals, respectively. Furthermore, this strategy was successfully implemented in dual-mode imaging of rheumatoid arthritis mice. Importantly, this study emphasizes a new design guideline for the maximization of fluorescence-photoacoustic signals by using dual-wavelength-independent excitation.

Upregulation of Formaldehyde in Parkinson's Disease Found by a Near-Infrared Lysosome-Targeted Fluorescent Probe.

Parkinson's disease (PD) is one of the major neurodegenerative diseases caused by complex pathological processes. As a signal molecule, formaldehyde is closely linked to nervous systems, but the relationship between PD and formaldehyde levels remains largely unclear. We speculated that formaldehyde might be a potential biomarker for PD. To prove it, we constructed the first near-infrared (NIR) lysosome-targeted formaldehyde fluorescent probe (named NIR-Lyso-FA) to explore the relationship between formaldehyde and PD. The novel fluorescent probe achieves formaldehyde detection in vitro and in vivo, thanks to its excellent properties such as NIR emission, large Stokes shift, and fast response to formaldehyde. Crucially, utilizing the novel probe NIR-Lyso-FA, formaldehyde overexpression was discovered for the first time in cellular, zebrafish, and mouse PD models, supporting our guess that formaldehyde can function as a possible biomarker for PD. We anticipate that this finding will offer insightful information for PD pathophysiology, diagnosis, medication development, and treatment.

Visualization of Lysosomal Dynamics during Autophagy by Fluorescent Probe.

Lysosomes are one of the important organelles within cells, and their dynamic movement processes are associated with many biological events. Therefore, real-time monitoring of lysosomal dynamics processes has far-reaching implications. A lysosome-targeted fluorescent probe N(CH2)3-BD-PZ is proposed for real-time monitoring of lysosomal kinetic motility. Using this probe, the dynamic process of lysosomes under starvation induction was successfully explored through fluorescence imaging. Importantly, we observed a new pattern of lysosomal dynamic movement, in which an irregular lysosome was slowly cleaved into two different-sized touching lysosomes and then fused to form a new round lysosome. This research provides a powerful fluorescence tool to understand the dynamic motility of intracellular lysosomes under fluorescence imaging.

Self-assembly of molecular beacons through metal ion coordination for fluorescence imaging of miRNA in living cells.

Fluorescence nanosensors based on functional nucleic acids have been explored as a powerful sensing platform for disease-relevant miRNAs. This work developed a new hybrid nanosensor (Zr-B) through coordination-driven self-assembly of Zr ions and beacons. The prepared nanosensor exhibited high loading efficiency of beacons and could achieve sensitive and specific detection for miRNAs. The hybrid nanosensor could transfer beacons into living cells efficiently and maintain high stability and biocompatibility in the biological environment, achieving effective miRNA fluorescence imaging in living cells. Therefore, the resultant nanosensor holds potential for applications in disease diagnostics.

A novel 1,8-naphthalimide-based fluorescent chemosensor for the detection of HSA in living cells.

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8-naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells.

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity.

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1. LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.