RESUMO
Mitochondria (MT) and the endoplasmic reticulum (ER) maintain lipid and calcium homeostasis through membrane contacts, particularly MT-ER contacts (MERCs), spanning distances from 10 to 50 nm. However, the variation of different distance ranges and the metabolic factors influencing this variation remain poorly understood. This study employed microfluidic chip-based super-resolution microscopy in conjunction with a Moore-Neighbor tracing-incorporated organelle proximity analysis algorithm. This approach enabled precise three-dimensional localization of single-fluorescence protein molecules within narrow and irregular membrane proximities. It achieved lateral localization precision of less than 20 nm, resulting in a minimum MERC distance of approximately 8 nm in spatial and mean distances across multiple threshold ranges. Additionally, we demonstrated that the MERC distance variation was correlated with MT size rather than ER width. The proportion of each distance range varied significantly after the stimuli. Free cholesterol showed a negative correlation with various distances, while distances of 10-30 nm were associated with glucose, glutamine, and pyruvic acid. Furthermore, the 30-40 nm range was influenced by citric acid. These results underscore the role of advanced subcellular organelle analysis in elucidating the single-molecule behavior and organelle morphology in single-cell studies.
Assuntos
Retículo Endoplasmático , Mitocôndrias , Análise de Célula Única , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Microscopia de Fluorescência/métodos , Células HeLaRESUMO
Visualization of the mitochondrial state is crucial for tracking cell life processes and diagnosing disease, while fluorescent probes that can accurately assess mitochondrial status are currently scarce. Herein, a fluorescent probe named "SYN" was designed and prepared, which can target mitochondria via the mitochondrial membrane potential. Upon pathology or external stimulation, SYN can be released from the mitochondria and accumulate in the nucleolus to monitor the status of mitochondria. During this process, the brightness of the nucleolus can then serve as an indicator of mitochondrial damage. SYN has demonstrated excellent photostability in live cells as well as an extremely inert fluorescence response to bioactive molecules and the physiological pH environment of live cells. Spectroscopic titration and molecular docking studies have revealed that SYN can be lit up in nucleoli due to the high viscosity of the nucleus and the strong electrostatic interaction with the phosphate backbone of RNA. This probe is expected to be an exceptional tool based on its excellent imaging properties for tracking mitochondrial state in live cells.
Assuntos
Nucléolo Celular , Corantes Fluorescentes , Mitocôndrias , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Corantes Fluorescentes/química , Nucléolo Celular/metabolismo , Células HeLa , Simulação de Acoplamento Molecular , Imagem Óptica , Potencial da Membrana MitocondrialRESUMO
Hydrogen peroxide (H2O2) overexpressed in mitochondria has been regarded as a key biomarker in the pathological processes of various diseases. However, there is currently a lack of suitable mitochondria-targetable near-infrared (NIR) probes for the visualization of H2O2 in multiple diseases, such as PM2.5 exposure-induced lung injury, hepatic ischemia-reperfusion injury (HIRI), nonalcoholic fatty liver (NAFL), hepatic fibrosis (HF), and malignant tumor tissues containing clinical cancer patient samples. Herein, we conceived a novel NIR fluorescent probe (HCy-H2O2) by introducing pentafluorobenzenesulfonyl as a H2O2 sensing unit into the NIR hemicyanine platform. HCy-H2O2 exhibits good sensitivity and selectivity toward H2O2, accompanied by a remarkable "turn-on" fluorescence signal at 720 nm. Meanwhile, HCy-H2O2 has stable mitochondria-targetable ability and permits monitoring of the up-generated H2O2 level during mitophagy. Furthermore, using HCy-H2O2, we have successfully observed an overproduced mitochondrial H2O2 in ambient PM2.5 exposure-induced lung injury, HIRI, NAFL, and HF models through NIR fluorescence imaging. Significantly, the visualization of H2O2 has been achieved in both tumor-bear mice as well as surgical specimens of cancer patients, making HCy-H2O2 a promising tool for cancer diagnosis and imaging-guided surgery.
Assuntos
Corantes Fluorescentes , Peróxido de Hidrogênio , Mitocôndrias , Imagem Óptica , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Peróxido de Hidrogênio/metabolismo , Animais , Mitocôndrias/metabolismo , Mitocôndrias/química , Camundongos , Humanos , Lesão Pulmonar/diagnóstico por imagem , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Raios InfravermelhosRESUMO
The level of sulfur dioxide (SO2) and viscosity in mitochondria play vital roles in various physiological and pathological processes. Abnormalities in mitochondrial SO2 and viscosity are closely associated with numerous biological diseases. It is of great significance to develop novel fluorescence probes for simultaneous detection of SO2 and viscosity within mitochondria. Herein, we have developed a water-soluble, mitochondrial-targeted and near-infrared fluorescent probe, CMBT, for the simultaneous detection of SO2 and viscosity. The probe CMBT incorporates benzothiazolium salt as a mitochondrial targeting moiety and 7-diethylaminocoumarin as a rotor for viscosity detection, respectively. Based on the prompt reaction between nucleophilic HSO3-/SO32- and the backbone of the benzothiazolium salt derivative, probe CMBT displayed high sensitivity and selectivity toward SO2 with a limit of detection as low as 0.17 µM. As viscosity increased, the twisted intramolecular charge transfer (TICT) process was restricted, resulting in fluorescence emission enhancement at 690 nm. Moreover, probe CMBT demonstrated exceptional mitochondrial targeting ability and was successfully employed to image variations of SO2 and viscosity in living cells and mice. The work highlights the great potential of the probe as a convenient tool for revealing the relationship between SO2 and viscosity in biological systems.
Assuntos
Corantes Fluorescentes , Mitocôndrias , Dióxido de Enxofre , Dióxido de Enxofre/análise , Dióxido de Enxofre/química , Corantes Fluorescentes/química , Animais , Mitocôndrias/química , Mitocôndrias/metabolismo , Viscosidade , Camundongos , Humanos , Imagem Óptica/métodos , Células HeLa , Limite de DetecçãoRESUMO
Cysteine (Cys) plays an indispensable role as an antioxidant in the maintenance of bioredox homeostasis. We have constructed an efficient fluorescent probe Mito-Cys based on the binding of indole and naphthol. The acrylic ester group serves as a recognition switch for specific detection of Cys, which undergoes Michael addition and intramolecular cyclization reactions, thereby ensuring the chemical kinetics priority of Cys compared to other biothiols. The probe has good water solubility, large Stokes shift (137 nm), with a detection limit of 21.81 nM. In addition, cell imaging experiments have shown that the probe has excellent mitochondrial targeting ability (R = 0.902). The probe can distinguish between Cys, homocysteine (Hcy) and glutathione (GSH), and can detect Cys specifically and quickly (100 s) to ensure accurate quantitative analysis of Cys changes in cells. More importantly, the probe confirms that ferroptosis inducing factors trigger thiol starvation in mitochondria, which helps to gain a deeper understanding of the physiological and pathological functions related to Cys and ferroptosis.
Assuntos
Cisteína , Corantes Fluorescentes , Mitocôndrias , Peixe-Zebra , Peixe-Zebra/metabolismo , Cisteína/metabolismo , Cisteína/química , Mitocôndrias/metabolismo , Mitocôndrias/química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Animais , Indóis/química , Indóis/metabolismo , Imagem Óptica , Estrutura Molecular , Naftóis/química , Naftóis/síntese química , Naftóis/metabolismoRESUMO
Neutral rhodol-based red emitters are shown to efficiently localize in mitochondria, as demonstrated by confocal microscopy and co-localization studies. A simple model is proposed to explain the localization mechanism of neutral molecules. The model takes into account the strong coupling between the molecular dipole moment and the electric field of the inner mitochondrial membrane.
Assuntos
Corantes Fluorescentes , Mitocôndrias , Mitocôndrias/metabolismo , Mitocôndrias/química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Microscopia Confocal , Xantonas/química , Estrutura Molecular , Células HeLaRESUMO
The heptamethine cyanine dyes are one kind of promising near-infrared (NIR) compounds, holding great potential in both diagnostic and therapeutic regions. Remolding such structures to realize detection of unclarified biotargets or interfering with them seems to be important in the field of chemical biology. In this study, we developed a fluorescent ligand (IR1) targeting mitochondrial G-quadruplexes (mitoG4s) by a slight variation on the typical NIR scaffold (IR780). This ligand could be applied for sensing mitoG4s by fluorescence, making it different from the unmodified dye whose fluorescence was quenched by mitoG4s. Then, IR1 was demonstrated to accumulate in the mitochondria through a mitochondrial membrane potential (MMP) dependent manner. Some of IR1 then bound to mitoG4s, causing mtDNA loss and mitochondrial dysfunction, which thereby triggered PANoptosis, including apoptosis, autophagy and pyroptosis. To the best of our knowledge, IR1 was the first NIR fluorescent ligand with emission centered at above 800 nm for mitoG4s, and the first example causing PANoptosis among the reported mitoG4-targeted ligands.
Assuntos
Carbocianinas , Corantes Fluorescentes , Quadruplex G , Mitocôndrias , Humanos , Apoptose/efeitos dos fármacos , Carbocianinas/química , DNA Mitocondrial/química , DNA Mitocondrial/genética , Corantes Fluorescentes/química , Ligantes , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/química , Mitocôndrias/metabolismo , Células HCT116 , Variações do Número de Cópias de DNARESUMO
The inner mitochondrial membrane (IMM) undergoes dynamic morphological changes, which are crucial for the maintenance of mitochondrial functions as well as cell survival. As the dynamics of the membrane are governed by its lipid components, a fluorescent probe that can sense spatiotemporal alterations in the lipid properties of the IMM over long periods of time is required to understand mitochondrial physiological functions in detail. Herein, we report a red-emissive IMM-labeling reagent with excellent photostability and sensitivity to its environment, which enables the visualization of the IMM ultrastructure using super-resolution microscopy as well as of the lipid heterogeneity based on the fluorescence lifetime at the single mitochondrion level. Combining the probe and fluorescence lifetime imaging microscopy (FLIM) showed that peroxidation of unsaturated lipids in the IMM by reactive oxygen species caused an increase in the membrane order, which took place prior to mitochondrial swelling.
Assuntos
Corantes Fluorescentes , Membranas Mitocondriais , Imagem Óptica , Corantes Fluorescentes/química , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/química , Humanos , Lipídeos/química , Microscopia de Fluorescência , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/análise , Células HeLa , Mitocôndrias/metabolismo , Mitocôndrias/químicaRESUMO
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.
Assuntos
Corantes Fluorescentes , Gotículas Lipídicas , Mitocôndrias , Prótons , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/química , Mitocôndrias/metabolismo , Mitocôndrias/química , Corantes Fluorescentes/química , Humanos , Células HeLa , AutofagiaRESUMO
Artificial organelles serve as functional counterparts to natural organelles, which are primarily employed to artificially replicate, restore, or enhance cellular functions. While most artificial organelles exhibit basic functions, we diverge from this norm by utilizing poly(ferrocenylmethylethylthiocarboxypropylsilane) microcapsules (PFC MCs) to construct multifunctional artificial organelles through water/oil interfacial self-assembly. Within these PFC MCs, enzymatic cascades are induced through active molecular exchange across the membrane to mimic the functions of enzymes in mitochondria. We harness the inherent redox properties of the PFC polymer, which forms the membrane, to facilitate in-situ redox reactions similar to those supported by the inner membrane of natural mitochondria. Subsequent studies have demonstrated the interaction between PFC MCs and living cell including extended lifespans within various cell types. We anticipate that functional PFC MCs have the potential to serve as innovative platforms for organelle mimics capable of executing specific cellular functions.
Assuntos
Compostos Ferrosos , Oxirredução , Silanos , Compostos Ferrosos/química , Silanos/química , Organelas/química , Organelas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Biomimética/métodos , Materiais Biomiméticos/química , Cápsulas/química , Polímeros/químicaRESUMO
Fluorescent labeling is a widely used method for protein detection and fluorescence imaging. A solvatochromic and fluorogenic molecular rotor DASPBCl was developed for covalent protein labeling in solution and SDS-PAGE, and also for stable mitochondria labeling and fluorescence imaging. The dye DASPBCl consisted of a 4-(N,N-dimethylamino)phenyl moiety as the electron donor and a positively charged N-benzylpyridinium moiety as the electron acceptor. A benzyl chloride group was introduced into the pyridine moiety for covalent labeling of thiol in proteins. When the fluorescent dye DASPBCl is covalently labeled to the thiol of proteins, significantly enhanced fluorescence was obtained, which is attributed to the polarity sensitivity caused solvatochromic effect from the hydrophobic protein structure and the viscosity sensitivity caused fluorogenic effect from the restriction of single bond rotation. DASPBCl exhibits high sensitivity and good linear response for protein detection in SDS-PAGE analysis with both the pre-staining method and post-staining method. DASPBCl was also successfully used for covalently protein-anchored fluorescence imaging of mitochondria in living cells.
Assuntos
Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Mitocôndrias , Compostos de Sulfidrila , Corantes Fluorescentes/química , Mitocôndrias/química , Mitocôndrias/metabolismo , Humanos , Eletroforese em Gel de Poliacrilamida/métodos , Compostos de Sulfidrila/química , Compostos de Sulfidrila/análise , Imagem Óptica/métodos , Células HeLa , Coloração e Rotulagem/métodos , Proteínas/química , Proteínas/análiseRESUMO
Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.
Assuntos
Mitocôndrias , Animais , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Técnicas de Cultura de Células/métodosRESUMO
Mitochondria play a pivotal part in ATP energy production through oxidative phosphorylation, which occurs within the inner membrane through a series of respiratory complexes1-4. Despite extensive in vitro structural studies, determining the atomic details of their molecular mechanisms in physiological states remains a major challenge, primarily because of loss of the native environment during purification. Here we directly image porcine mitochondria using an in situ cryo-electron microscopy approach. This enables us to determine the structures of various high-order assemblies of respiratory supercomplexes in their native states. We identify four main supercomplex organizations: I1III2IV1, I1III2IV2, I2III2IV2 and I2III4IV2, which potentially expand into higher-order arrays on the inner membranes. These diverse supercomplexes are largely formed by 'protein-lipids-protein' interactions, which in turn have a substantial impact on the local geometry of the surrounding membranes. Our in situ structures also capture numerous reactive intermediates within these respiratory supercomplexes, shedding light on the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparison of supercomplexes from mitochondria treated under different conditions indicates a possible correlation between conformational states of complexes I and III, probably in response to environmental changes. By preserving the native membrane environment, our approach enables structural studies of mitochondrial respiratory supercomplexes in reaction at high resolution across multiple scales, from atomic-level details to the broader subcellular context.
Assuntos
Respiração Celular , Complexo III da Cadeia de Transporte de Elétrons , Complexo I de Transporte de Elétrons , Mitocôndrias , Animais , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/ultraestrutura , Modelos Moleculares , Fosforilação Oxidativa , Suínos , Ubiquinona/análogos & derivados , Ubiquinona/química , Ubiquinona/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismoRESUMO
A novel NIR fluorescent probe based on quinoline-conjugated benzo[cd]indol dual-salt for NADH was developed. This probe swiftly detects and responds sensitively to both endogenous and exogenous NADH alterations, enabling imaging of NADH fluctuations in type II diabetic and AD model cells.
Assuntos
Corantes Fluorescentes , Mitocôndrias , NAD , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , NAD/análise , NAD/química , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Quinolinas/química , Raios Infravermelhos , Imagem Óptica , Animais , Diabetes Mellitus Tipo 2RESUMO
SO2 derivatives play an important role in many metabolic processes, excessive ingestion of them can lead to serious complications of various diseases. In this work, a novel dual ratiometric NIR fluorescent probe XT-CHO based on ICT effect was synthesized for detecting SO2 derivative. In the design of the probe, the α, ß-unsaturated bond formed between benzopyran and coumarin was used as the reaction site for SO2, meanwhile, the extended π-conjugate system promoted maximum emission wavelength of the probe up to 708 nm. Notably, the probe exhibited high selectivity and sensitivity for detecting SO2, the limit of detection reached 2.13 nM and 58.5 nM in fluorescence spectra and UV-Vis absorption spectra, respectively. The reaction mechanism of SO2 and XT-CHO had been verified by 1H NMR, ESI-MS spectra and DFT calculation. Moreover, the probe was successfully applied in detecting endogenous and exogenous SO2 in living cells and proved possessed the mitochondrial targeted ability.
Assuntos
Corantes Fluorescentes , Mitocôndrias , Dióxido de Enxofre , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Dióxido de Enxofre/análise , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Espectrometria de Fluorescência , Células HeLa , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Cumarínicos/química , Cumarínicos/síntese química , Limite de Detecção , Teoria da Densidade Funcional , Imagem ÓpticaRESUMO
Many diseases in the human body are related to the overexpression of viscosity and sulfur dioxide. Therefore, it is essential to develop rapid and sensitive fluorescent probes to detect viscosity and sulfur dioxide. In the present work, we developed a dual-response fluorescent probe (ES) for efficient detection of viscosity and sulfur dioxide while targeting mitochondria well. The probe generates intramolecular charge transfer by pushing and pulling the electron-electron system, and the ICT effect is destroyed and the fluorescence quenched upon reaction with sulfite. The rotation of the molecule is inhibited in the high-viscosity system, producing a bright red light. In addition, the probe has good biocompatibility and can be used to detect sulfite in cells, zebrafish and mice, as well as upregulation of viscosity in LPS-induced inflammation models. We expect that the dual response fluorescent probe ES will be able to detect viscosity and sulfite efficiently, providing an effective means of detecting viscosity and sulfite-related diseases.
Assuntos
Corantes Fluorescentes , Inflamação , Mitocôndrias , Sulfitos , Peixe-Zebra , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Animais , Sulfitos/química , Sulfitos/análise , Viscosidade , Mitocôndrias/metabolismo , Mitocôndrias/química , Camundongos , Humanos , Inflamação/induzido quimicamente , Dióxido de Enxofre/análise , Dióxido de Enxofre/química , Lipopolissacarídeos , Células RAW 264.7 , Imagem Óptica/métodosRESUMO
Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.
Assuntos
Cobre , Corantes Fluorescentes , Potencial da Membrana Mitocondrial , Agregados Proteicos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Cobre/química , Cobre/metabolismo , Humanos , Corantes Fluorescentes/química , Mitocôndrias/metabolismo , Mitocôndrias/química , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/química , Células HeLaRESUMO
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 (â¼690 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.
Assuntos
Ferroptose , Mitocôndrias , Mitofagia , Mitocôndrias/química , Mitocôndrias/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Animais , Células COS , Chlorocebus aethiops , Viscosidade , Sobrevivência Celular , Elastina/genética , Elastina/metabolismoRESUMO
Viscosity, at the subcellular level, plays a crucial role as a physicochemical factor affecting microenvironment homeostasis. Abnormal changes in mitochondrial viscosity often lead to various diseases in the organism. Based on the twisted intramolecular charge transfer mechanism, four hemicyanine dye fluorescent probes (HT-SA, HT-SA-S, HT-Bzh, and HT-NA) were designed and synthesized for viscosity response. The single bond between the nitrogen-containing heterocycle and the carbon-carbon double in the structure of the probe bond served as the viscosity response site. Finally, the probe HT-Bzh was screened as the optimal mitochondrial viscosity probe according to its responsiveness, targeting, and interference resistance. The fluorescence intensity of the probe HT-Bzh increased 22-fold when the viscosity was increased from 13.75 to 811.2 cP. In summary, all four viscosity probes we have developed can be used in different applications depending on the external environment, providing a valuable reference for the design of potential tools to address viscosity monitoring in biological systems.
Assuntos
Carbocianinas , Corantes Fluorescentes , Mitocôndrias , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Viscosidade , Carbocianinas/química , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Células HeLa , Estrutura Molecular , Imagem ÓpticaRESUMO
Nitric oxide (NO) is a crucial signal molecule closely linked to the biological immune response, especially in macrophage polarization. When activated, macrophages enter a pro-inflammatory state and produce NO, a marker for the M1 phenotype. In contrast, the anti-inflammatory M2 phenotype does not produce NO. We developed a mitochondria-targeted two-photon iridium-based complex (Ir-ImNO) probe that can detect endogenous NO and monitor macrophages' different immune response states using various imaging techniques, such as one- and two-photon phosphorescence imaging and phosphorescence lifetime imaging. Ir-ImNO was used to monitor the immune activation of macrophages in mice. This technology aims to provide a clear and comprehensive visualization of macrophage immune responses.