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[Methods in Molecular Biology] Calcium Signalling Volume 1925 (Methods and Protocols) || High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels
摘要： Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.
关键词： Cytosol,Calcium probes,Calcium,High-throughput screening,Aequorin,Mitochondria,ER
更新于2025-11-21 11:20:42
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Enhancing magnetic resonance/photoluminescence imaging-guided photodynamic therapy by multiple pathways
摘要： Mitochondria, which are a major source of adenosine triphosphate (ATP) and apoptosis regulators, are the key organelles that promote tumor cell proliferation, and their dysfunction affects tumor cell behavior. Additionally, mitochondria have been shown to play a central role in the biosynthesis of protoporphyrin IX (PpIX), which is a widely used photosensitizer that has been used for tumor detection, monitoring and photodynamic therapy. Nevertheless, photosensitizers administrated exogenously are often restricted by limited bioavailability. δ-Aminolevulinic acid (δ-ALA) is a naturally occurring delta amino acid that can be converted in situ to PpIX via the heme biosynthetic pathway in mitochondria. Because δ-ALA is the precursor for PpIX, δ-ALA-based photodynamic therapy (PDT) shows promise in treating cancer. However, the accumulation of δ-ALA within endosomal system limits the production of PpIX and eventually impedes its effectiveness. Theranostic nanoparticles (NPs) capable of endosomal escape are expected to optimize the endogenous biosynthetic yield. In this study, δ-ALA was improved with triphenylphosphonium cation (TPP+), a high net position cation in endosomal escape and as a mitochondria-targeting ligand, and was further modified with bovine serum albumin stabilized manganese dioxide (MnO2). The tumor microenvironment (TME) responsive MnO2 in this system can elevate oxygen content to relieve hypoxia. Both enhanced photosensitizer yield and elevated oxygen contributing to the final therapeutic effect. Moreover, the enhancement of magnetic resonance imaging (MRI) (r1=5.410 s-1mM-1) stemming from the degradation of MnO2 by the TME could serve as a guide prior to treatment for accurate location, while in situ hysteretic photoluminescence imaging derived from PpIX can be utilize as a supervisor for the biomedical prognosis evaluation. This systematic design could broaden application and highlight the considerable therapeutic promise of PDT.
关键词： dual-imaging nanoplatform,mitochondria,endogenously biosynthetic photosensitizer
更新于2025-11-21 11:08:12
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Visualizing Nitric Oxide in Mitochondria and Lysosomes of Living Cells with N-Nitrosation of BODIPY-based Fluorescent Probes
摘要： Nitric oxide (NO), a ubiquitous gasotransmitter which plays critical roles in cardiovascular, nervous, and immune systems related diseases, is closely related in the physiological and pathological processes of mitochondria and lysosomes. Thus, monitoring NO in mitochondria or lysosomes is very meaningful for NO related chemical biology. Herein, we rationally designed four NO probes, BDP-NO, Mito-NO-T, Mito-NO and Lyso-NO, based on BODIPY dye substituted at meso position with 5-amino-2-methoxy-phenyl scaffold. These four probes all showed fast fluorescence off-on response toward NO with excellent selectivity and high sensitivity with the detection limit of BDP-NO to reach 5.7 nM. We introduced triphenylphosphonium and morpholine moieties onto BODIPY scaffold respectively to enable organelle-targetability. MTT and flow cytometry assay demonstrated that the probes exhibited low cytotoxicity, which was beneficial to the biological application in living cells. Confocal fluorescence microscopy experiments confirmed excellent mitochondria targeting for Mito-NO and lysosome-targeting with Lyso-NO for the detection of NO in living cells.
关键词： Fluorescent probes,Mitochondria-targeted,Lysosomes-targeted,BODIPY,Nitric oxide
更新于2025-11-21 11:08:12
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A mitochondria-targeted ratiometric fluorescent probe for detection of SO2 derivatives in living cells and in vivo
摘要： A new near-infrared (NIR) ?uorescent probe for colorimetric and ratiometric detection of SO2 derivatives was developed based on conjugated hybrid coumarin-hemicyanine. The probe can detect HSO3?/SO32? in HEPES bu?er (10 mM, pH 7.4, with 10% DMF, v/v) with a large emission shift (259 nm). Importantly, it was successfully used for ?uorescence imaging of endogenous bisul?te in BT-474 cells and zebra?sh.
关键词： Near-infrared ?uorescent probe,SO2 derivatives,Mitochondria-targeted,Ratiometric,Colorimetric
更新于2025-11-19 16:56:35
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Photostable Ratiometric Two-photon Fluorescent Probe for Visualizing Hydrogen Polysulfide in Mitochondria and Its application
摘要： Hydrogen polysulfide (H2Sn) has currently attracted much research interest because it not only plays important physiological function in many biological and health-related events, but also considered as a newfound potent signal transducer. Small-molecule based ratiometric fluorescent probes have advantages in sensitivity and bio-detections but such approaches that intentionally developed for H2Sn detection expected to be mitochondria-accessible are still lacking. In this work, due to that triphenylphosphine group introduced into the molecular scaffold of naphthalimide derivative, Mito-NRT-HP was successfully applied to visualize intracellular H2Sn in mitochondria with excellent aqueous solubility, super photobleaching resistance, favorable cellular membrane permeability and good biocompatibility. This one- and two-photon fluorescent probe with high selectivity and sensitivity (LOD = 0.01 μM) evinced 70-fold enhancement of fluorescence ratio (I546 nm/I478 nm) in the presence of H2Sn over other reactive sulfur species (RSS). The experimental results also give Mito-NRT-HP the potential for mapping the H2Sn distribution in mitochondria and evaluating the H2Sn roles in more biological processes and demonstrated the practical application possibility of Mito-NRT-HP in early diagnosis of LPS-induced acute organ injury.
关键词： mitochondria,fluorescent probe,hydrogen polysulfide,ratiometric,two-photon
更新于2025-11-14 15:29:11
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Suppression of Light-Induced Oxidative Stress in the Retina by Mitochondria-Targeted Antioxidant
摘要： Light-induced oxidation of lipids and proteins provokes retinal injuries and results in progression of degenerative retinal diseases, such as, for instance, iatrogenic photic maculopathies. Having accumulated over years retinal injuries contribute to development of age-related macular degeneration (AMD). Antioxidant treatment is regarded as a promising approach to protecting the retina from light damage and AMD. Here, we examine oxidative processes induced in rabbit retina by excessive light illumination with or without premedication using mitochondria-targeted antioxidant SkQ1 (10-(6’-plastoquinonyl)decyltriphenyl-phosphonium). The retinal extracts obtained from animals euthanized within 1–7 days post exposure were analyzed for H2O2, malondialdehyde (MDA), total antioxidant activity (AOA), and activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) using colorimetric and luminescence assays. Oxidation of visual arrestin was monitored by immunoblotting. The light exposure induced lipid peroxidation and H2O2 accumulation in the retinal cells. Unexpectedly, it prominently upregulated AOA in retinal extracts although SOD and GPx activities were compromised. These alterations were accompanied by accumulation of disulfide dimers of arrestin revealing oxidative stress in the photoreceptors. Premedication of the eyes with SkQ1 accelerated normalization of H2O2 levels and redox-status of lipids and proteins, contemporarily enhancing AOA and, likely, sustaining normal activity of GPx. Thus, SkQ1 protects the retina from light-induced oxidative stress and could be employed to suppress oxidative damage of proteins and lipids contributing to AMD.
关键词： SkQ1,superoxide dismutase,glutathione peroxidase,disulfide dimerization of proteins,visual arrestin,age-related macular degeneration,mitochondria-targeted antioxidant,antioxidant activity,light-induced retinal damage,oxidative stress
更新于2025-09-23 15:23:52
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Mitochondria-targeted ratiometric fluorescent probes for micropolarity and microviscosity and their applications
摘要： Ambient polarity and viscosity are two important parameters that influence many chemical and biological processes of biomolecules. In biological systems especially at the cellar level, polarity affects the interaction of many proteins and enzymes as well as the cell membrane permeability. Microenvironmental viscosity has a marked impact on some physiological processes such as cellular material transportation, interactions between biological macromolecules, diffusion of reactive substances. Abnormal changes in polarity and/or viscosity have been considered as vital causes or indicators for some diseases and malfunctions such as diabetes and Alzheimer's disease. Detection of microenvironmental polarity and/or viscosity has caused much interest in many scientific areas. Mitochondria are important organelles of energy production as well as major sites of aerobic respiration in eukaryotes. They are also involved in lots of significant processes including protein translocation, the synthesis of ATP, the transportation of metabolites, etc. The mitochondrial functions are closely linked with physical chemical properties of mitochondria, namely polarity, viscosity, pH and temperature. Therefore, monitoring the changes in mitochondrial polarity and/or viscosity is fruitful for understanding some important physiological processes. However, conventional viscometers and polarity detectors are not suitable for the measurements at the cellar level. It is highly desired to develop new techniques for fast quantifying cellar polarity and viscosity. Fluorescence-based techniques have been widely applied in many scientific researches due to the merits of good accuracy, extremely high sensitivity, temporal and spatial resolution and especially bioimaging in living cells. Numerous polarity-sensitive sensors have been developed for detection of cellar polarity based on intramolecular charge transfer (ICT) mechanism. For viscosity tests, 'molecular rotors' have been proved to be promising probes for their excellent photophysical properties of ratiometric fluorescence and/or fluorescence lifetime imaging. Nevertheless, probe possesses features of both ICT and molecular rotor and could monitor polarity and viscosity simultaneously has rarely been reported. Coumarin is a well-known polarity-sensitive fluorophore, and many coumarin-based probes have been constructed for polarity. Normally, the structural characteristic of a molecular rotor is that a steric group covalently linked to a planar fluorophore with a single/double bond as the connecting rotation shaft. In view of the above points, we envisioned that if a steric group linked to a polarity-sensitive fluorophore, it may detect polarity and viscosity simultaneously. Herein, two compounds YYH1 and YYH2 with coumarin as the fluorophore and N-methyl indole as the steric group were selected to quantify microenvironmental polarity and viscosity. Compared to those with similar fluorophore reported in the references, our probes were more sensitive to polarity and/or viscosity and have longer emission wavelengths (λem = 685 nm for YYH2). Furthermore, the cationic characteristics of both compounds endows them locating in mitochondria in living cells. With compounds YYH1 and YYH2 (NMR and mass spectra are shown in Figs. S1 and S2 in Supporting information) in hand, we first investigated their photophysical properties in phosphate buffered solution (PBS). Fig. S3 (Supporting information) demonstrates that both compounds have one absorption and two emission peaks in PBS. The emission peaks of shorter wavelengths could be ascribed to the part without indole moiety. The red emission bands are resulted from the whole π-conjugation system, which are sensitive to the solvent viscosity. The emission (681 nm) and absorption (597 nm) maxima of YYH2 are at the red wavelength region and about 30 nm longer than those of YYH1, which enables the probe to be potentially applied in biological systems. Considering the strong ICT characteristic from the aminocoumarin unit to the indole part, the solvent-dependent spectral properties were studied. Both compounds display solvatochromic absorption and emission spectra (Figs. S4 and S5 in Supporting information). The absorption maximum of YYH1 shifted from 628 nm in toluene to 565 nm in PBS, while the green emission moved from 462 nm to 478 nm in the same series of solvents. However, the red emission showed little change upon polarity variation (Fig. S6 in Supporting information). Good relationship between absorption/emission wavenumber and the solvent polarity parameters (ET) verifies the ICT characteristic of the green emission (Fig. S6). The polarities of cationic compounds YYH1 and YYH2 are greater in ground state than in excited state, therefore, the absorption wavelength decreased with increasing polarity. Nevertheless, the green emission wavelength increased with increasing polarity, suggesting that the indole cation section was not involved in the green emission. In 1,4-dioxane-water binary mixture, with increasing water content, the absorption peak shifted from 613 to 565 nm and 647 to 597 nm for YYH1 and YYH2, respectively (Fig. S7 and S8 in Supporting information). The emission wavelength of YYH1 shifted from 468 nm to 478 nm without obvious intensity change when the proportion of the polar solvent water increased from 10% to 20% (Fig. 1a). Further increasing water content resulted in significant decrease in fluorescence intensity of the green emission with no appreciable wavelength change, whereas the emission at 645 nm increased steadily, with an isoemission point at 598 nm (Fig. 1a). More than 150 nm wavelength difference between the two bands provides a ratiometric response. The ratio I490/I645 decreased dramatically from 27 to 0.5 with increasing water content from 20% to 100% suggesting that YYH1 could be a potential ratiometric fluorescent probe for microenvironmental polarity. In the case of YYH2, when water content changed from 0 to 40%, both the blue (515 nm) and red (685 nm) emissions increased; while they both decreased with further increment of water content (Figs. S8b and c). The fluorescence intensity ratio I515/I685 hardly changed with water content revealing that YYH2 is insensitive to polarity (Fig. 1b). Then the spectral responses of YYH1 and YYH2 toward arbitrary changes in the viscosity were tested to understand the viscosity effect. The experimental results illustrate that YYH1 and YYH2 respond to the viscosity in different ways. In glycol-water mixed system, the absorbance at 595 nm of YYH2 increased slightly accompanied with about 12 nm red-shift with increasing glycol content, which could be probably attributed to the slight decrease of the polarity (Fig. S9 in Supporting information). As shown in Fig. 2a, the red emission of YYH2 is more sensitive to solvent viscosity than the green one: with increasing solvent viscosity from 1.0 (water) to 19.9 cp (glycol), about 10-fold and 1.7-fold fluorescence enhancements respectively for the red and green emissions were observed. The enhancement in the green emission was possibly attributed to the variation in solution's polarity. Good linear relationship between log (I685/I515) and logη of the solvent reveals that YYH2 could be applied in ratiometric detection of the media viscosity. The quantitative relationship between the ratio of I685/I515 and the solvent viscosity η is well expressed by F?rster-Hoffmann equation: log(I685/I515) = C + xlogη (C = ?0.673, x = 0.634, R2 = 0.968), where C is a concentration and temperature-dependent constant and x is a dye-dependent constant. In addition, the fluorescence life time of YYH2 also became slightly longer with increasing solution viscosity (Fig. S10 in Supporting information): It was 0.3, 0.47 and 1.46 ns in water, 1:1 water-glycol and glycol, respectively. As for YYH1, about 8 nm blue-shift of the absorption maximum was found with increasing water content from 0 to 100%. The fluorescence intensities at 490 nm and 645 nm increased to almost the same extent (Fig. S11 in Supporting information), and the ratio of I645/I490 hardly changed with viscosity (Fig. 2b). From the above results, it is clear that YYH1 and YYH2 could be employed to detect local polarity and viscosity, respectively. To evaluate the biological application of the probes, we tested both compounds in living cells (Fig. 3). The localization of the probes in two living cell lines, L929 and MCF-7 cells, were determined by co-staining cells with mitrochondria-specific dye. The bright red fluorescent region with YYH1 (a2, b2) and green signals from Mitro Tracker Green FM (a1, b1) overlapped perfectly (a4, b4). A high Pearson correlation factor Pf of 0.91 for L929 cells (0.94 for MCF-7 cells) indicates that YYH1 is a mitrochondria-targeted dye. Similar results were obtained in the case of YYH2 (Figs. 3c and d) (Pf are 0.93 and 0.92 for L929 and MCF-7 cells, respectively), revealing that both probes could localize at mitrochondria. In living cells, proteins and some other biologically related substances are the main components and involved in every process within cells. The fluctuation of protein contents could affect cellar polarity or viscosity. To verify the effectiveness and practicability of the probes in realistic samples, the effects of some biological molecules on the spectral properties of YYH1 and YYH2 were measured. Table S1 (Supporting information) showed that both the ratio I490/I645 of YYH1 and the ratio I685/I515 of YYH2 became higher upon the addition of proteins (Fig. S12-S13 in Supporting information), revealing that the presence of proteins increased the microenvironmental viscosity and decreased the microenvironmental polarity of the probes. Furthermore, it is obvious that the viscosity and polarity varied with proteins (Table S1 in Supporting information), which means that the difference in protein content and/or protein species could cause the change in cellar viscosity and polarity. It is reported that probes YYH1 and YYH2 exhibited spectral responses toward sulfite. Other relevant species except for peroxynitrite hardly influence the photophysical properties of the probes. Therefore, the presence of sulfite or peroxynitrite could interfere with bioimaging to some extent. However, it is difficult for detection of peroxynitrite in living cells by traditional analytical methods due to its short half-life under typical physiological conditions. Sulfite content in living cells is relatively low because it could easily be oxidized to sulfate by sulfite oxidase. The interference from these ions could be neglected. On the other hand, the presence of sulfite resulted in the same variation tendency of Iblue/Ired for both probes. However, the change trends of Iblue/Ired for YYH1 and YYH2 caused by polarity/viscosity were quite different. Therefore, interference from sulfite could be distinguished by using both probes for bioimaging. Next, YYH1 and YYH2 were employed to estimate the polarity and viscosity in living cells, respectively. After MCF-7 and L929 cells incubated with YYH1 (5 μmol/L) for 30 min, blue and red imaging were collected. Strong intracellular blue fluorescence and weak intracellular red fluorescence were observed in both cells. Considering that the ratio of I490/I645 (i.e. I645/I490 in Fig. 2b) hardly changed with solvent viscosity, the ratio of Iblue/Ired may indicate that the mitochondrial polarity in L929 cells is smaller than that in MCF-7 cells (Fig. 4). After incubated with YYH2 (5 μmol/L) for 30 min, brighter intracellular green fluorescence was observed in MCF-7 cells, while stronger intracellular red fluorescence was observed in L929 cells (Fig. 5). The ratio of Ired/Igreen suggests that the mitochondrial viscosity in L929 cells is larger than that in MCF-7 cells. In summary, we have synthesized two mitochondria-targeted ratiometric fluorescent probes YYH1 and YYH2 for quantification of micropolarity and microviscosity, respectively. Both probes were constructed with environment-sensitive coumarin and N-methyl indole linked through a C-C bridge. YYH1 responded to the polarity, while YYH2 responded to the viscosity sensitively. Therefore, they were used for ratiometric fluorescent detection of the mitochondrial polarity and viscosity respectively. This work may provide some ideas for design of polarity and/or viscosity fluorescent probes for bioimaging living cells.
关键词： Mitochondria,Microviscosity,Micropolarity,Fluorescent probe
更新于2025-09-23 15:23:52
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A novel “turn-on” mitochondria-targeting near-infrared fluorescent probe for determination and bioimaging cellular hydrogen sulfide
摘要： Hydrogen sulfide (H2S) has been regarded as an important gas transmitter playing vital role in cytoprotective processes and redox signaling. It is very meaningful to monitor and analyze it in biosystem for obtaining important physiological and pathological information. Despite numerous fluorescent probes for cellular H2S have been reported in past decades, only a few have capability to detect mitochondrial H2S with near-infrared (NIR) emission. Therefore, a new mitochondria-targeting NIR fluorescent probe (Mito-NSH) for detection of cellular H2S was developed by introducing 2,4-dinitrophenyl ether into a novel dye (Mito-NOH). A large "turn-on" NIR fluorescence response was obtained due to thiolysis of ether to hydroxyl group when Mito-NSH was treated with NaHS. Moreover, Mito-NSH could quantitatively detect H2S at concentration ranging from 0 to 30 μM with a detection limit of 68.2 nM, and it exerts some superior optical properties, such as large stokes shift (107 nm), highly selectively mitochondria location, fast response and high selectivity to H2S. More impressively, it was successfully applied to imaging exogenous and endogenously generated H2S in living HeLa cells via confocal fluorescence microscopy.
关键词： Fast response,NIR fluorescent probe,"Turn-on" response,Hydrogen sulfide,Mitochondria-targeting
更新于2025-09-23 15:23:52
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Mitochondria-targeted and FRET based ratiometric fluorescent probe for SO2 and its cell imaging
摘要： SO2 can react with water to form sulphite (SO3 2-) and bisulphite (HSO3 -) ions, after it is inhaled into the body. Aberrant levels of SO2 and its derivatives in cells are related with a variety of diseases. So, it is important to monitor the level of SO2 and its derivatives in biological systems. Up to now, few probes have been reported for ratiometric detection of bisulfites in mitochondria. And the response speed of most reported probes were slow. It limited their application in real time detection of SO2. Here, using benzopyrylium as energy acceptor and mitochondria-targeted group, a FRET fluorescent probe CBP for SO2 was developed. CBP can detect SO2 with excellent selectivity over various analytes, including glutathione, homocysteine, cysteine and some common reactive oxygen species. The recognition process is accompanied by the color of the solution changing from purple to yellow, which allows “naked eye” detection for SO2. Its response is very fast (<1 min) and the detection limit is low (17.7 nM). In addition, probe CBP can be located in mitochondria and successfully used for the imaging of intracellular SO2 in EC-109 cells.
关键词： Fluorescence,Cell imaging,FRET,Ratiometric,Mitochondria
更新于2025-09-23 15:23:52
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Interference-free surface-enhanced Raman scattering nanosensor for imaging and dynamic monitoring of reactive oxygen species in mitochondria during photothermal therapy
摘要： Highly reliable detection, imaging, and monitoring of reactive oxygen species (ROS) at subcellular organelles are critical for understanding the biological roles of ROS and learning the pathogenesis of some diseases. In this study, we presented an interference-free surface-enhanced Raman scattering (SERS)-active nanoprobe for the intracellular ROS detection. This nanoprobe was designed as an Au core-Ag shell nanoparticle (Au@Ag NPs) with a SERS reporter (4-mercaptobenzonitrile) resided in the inner of the core-shell. Intracellular ROS is able to etch the Ag shell and dramatically decreases the SERS intensity of the SERS reporters. It is worth mentioning that the Raman band of the reporter we used in this study locate in the cellular Raman-silent region (1800?2800 cm?1), which eliminates interference possibility from cellular molecules. The shell can also further protect the reporters from the interference of mediums. We detected ROS at subcellular organelle level, e.g. mitochondria, by modifying the surface of the nanoprobes with a mitochondria-targeting peptide. And for the first time, the SERS-based monitoring of ROS at mitochondria during the photothermal therapy process was validated and the profiles of the ROS distribution were disclosed by SERS mapping.
关键词： ROS,mitochondria,SERS,PTT,interference-free
更新于2025-09-23 15:23:52