A ratiometric fluorescent probe for assessing mitochondrial phospholipid peroxidation within living cells

Mitochondrial oxidative damage contributes to a wide range of pathologies, and lipid peroxidation of the mitochondrial inner membrane is a major component of this disruption. However, despite its importance, there are no methods to assess mitochondrial lipid peroxidation within cells specifically. To address this unmet need we have developed a ratiometric, fluorescent, mitochondria-targeted lipid peroxidation probe, MitoPerOx. This compound is derived from the C11-BODIPY(581/591) probe, which contains a boron dipyromethane difluoride (BODIPY) fluorophore conjugated via a dienyl link to a phenyl group. In response to lipid peroxidation the fluorescence emission maximum shifts from ～590 to ～520nm. To target this probe to the matrix-facing surface of the mitochondrial inner membrane we attached a triphenylphosphonium lipophilic cation, which leads to its selective uptake into mitochondria in cells, driven by the mitochondrial membrane potential. Here we report on the development and characterization of MitoPerOx. We found that MitoPerOx was taken up very rapidly into mitochondria within cells, where it responded to changes in mitochondrial lipid peroxidation that could be measured by fluorimetry, confocal microscopy, and epifluorescence live cell imaging. Importantly, the peroxidation-sensitive change in fluorescence at 520nm relative to that at 590nm enabled the use of the probe as a ratiometric fluorescent probe, greatly facilitating assessment of mitochondrial lipid peroxidation in cells.     

A Novel Method for Continuous Determination of the Intracellular pH in Bacteria with the Internally Conjugated Fluorescent Probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester

A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.     

A dual-emission fluorescent probe for discriminating cysteine from homocysteine and glutathione in living cells and zebrafish models

Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and homocysteine (Hcy). In this study, a simple two-emission turn-on fluorescent biothiol probe (ICN-NBD) was rationally designed and synthesized through a facile ether bond linking 7-nitro-1,2,3-benzoxadiazole (NBD) and phenanthroimidazole containing a cyano tail. The probe in the presence of Cys elicited two fluorescence responses at 470 nm and 550 nm under excitation at 365 nm and 480 nm, respectively, because of the concomitant generation of both the fluorophore and NBD-N-Cys. In contrast, addition of Hcy and glutathione (GSH) could result in only a blue fluorescence enhancement at 470 nm. which was reasonably attributed to rearrangement from NBD-S-Hcy/GSH to NBD-N-Hcy/GSH as a result of geometrical constraints or solvent effects. Therefore, the fluorescent probe with the NBD scaffold could detect biothiols and simultaneously discriminate Cys from Hcy/GSH in both blue and green channels. The probe has been successfully applied for visualizing biothiols in living cells and zebrafish.     

A ratiometric fluorescent probe for bisulphite anion,employing intramolecular charge transfer

4‐(1H‐benzimidazol‐2‐yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisulphite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol, an aldehyde–bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluorescence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe for bisulphite is in the 2.0–200 µmol/L concentration range and the detection limit is 0.4 µmol/L. Probe 1 produces a ratiometric fluorescent response to bisulphite with a marked emission wavelength shift (130 nm) and displays high selectivity for bisulphite over other anions. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

A DIE responsive NIR-fluorescent cell membrane probe

It is challenging to achieve selective off to on modulation of the emissive state of a fluorophore within a complex and heterogeneous cellular environment. Herein we show that the dis-assembly of a non-fluorescent aggregate to produce individual fluorescent molecules, termed disaggregation induced emission (DIE), can be utilised to achieve this goal with an amphiphilic BF₂-azadipyrromethene (NIR-AZA) probe. Optical near-infrared properties of the NIR-AZA probe used in this study include absorption and emission maxima at 700 and 726 nm respectively when in the emissive non-aggregated state. Key to the success of the probe is the bis-sulfonic acid substitution of the NIR-AZA fluorophore, which is atypical for membrane probes as it does not contain zwitterionic lipid substituents. The aggregation/disaggregation properties of the NIR-fluorophore have been investigated in model surfactant and synthetic liposomal systems and shown to be emissive responsive to both. Real-time live cell imaging experiments in HeLa Kyoto and MC3T3 cells showed a rapid switch on of emission specific to the plasma membrane of viable and apoptotic cells attributable to a disaggregation-induced emission of the probe. Image analysis software confirmed localisation of fluorescence to the plasma membrane. Cell membrane staining was also effective for formaldehyde fixed cells, with staining possible either before or after fixation. This study adds new and important findings to recent developments of DIE responsive probes and further applications of this controllable emission-switching event are anticipated.     

A laser spectrofluorimeter for studies of cultured cells on the growth surface.

We have developed a new method for direct measurement of fluorescent probes associated with undisturbed monolayers of cultured fibroblasts. A helium-cadmium continuous wave laser produced excitation light at 325 nm to illuminate the cell monolayer on the front inner surface of a quartz sample tube. The emitted light from the cell monolayer passed through a scanning monochrometer to a low noise photomultiplier tube and was amplified with a photon-counting system. The fluorescent probe, cholesteryl 4-(3′-pyrenyl)butanoate, was incorporated into normal human low density lipoproteins (LDL). The interaction between LDL containing the fluorescent probe and the cell surface of normal human fibroblasts was examined. The uptake of the fluorescent LDL was measured as a function of temperature, concentration, time, specificity, and ability to suppress 3-hydroxy-3-methyl-glutaryl-CoA reductase. In all respects, LDL containing the fluorescent probe and native LDL were comparable. Using this technique, cell-surface interactions can be studied in situ so that changes in structure and function caused by removal of the cells from the growth surface can be avoided.     

A novel colorimetric fluorescent probe for detecting hydrazine in living cells and zebrafish

Hydrazine (NH₂NH₂) is a highly toxic organic substance that poses a threat to human health. Monitoring hydrazine with high sensitivity and selectivity is very important. Here, a simple colorimetric fluorescent probe for hydrazine detection, which is a seminaphthorhodafluor derivative containing thiophene-2-carboxylic acid ester reaction site, was rationally constructed. The probe itself exhibits weak fluorescence. The fluorescence is significantly enhanced when hydrazine is added. The probe exhibited a broad linear range (0–1 mM) with satisfactory selectivity and sensitivity (limit of detection 36.4 μM), which turned out to be an excellent fluorescent probe for monitoring hydrazine. Additionally, the probe was used to track hydrazine in living cells and zebrafish with great success, and the detection performance was satisfying. These results proved that this type of fluorescent probe with the thiophene-2-carboxylic acid ester structure can detect hydrazine with higher selectivity and sensitivity.     

A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited‐state intramolecular proton transfer

A ratiometric fluorescent probe 2‐(benzimidazol‐2‐yl)phenyl phosphoric acid (1) for alkaline phosphatase (ALP) is designed and synthesized. The method employs the modulation of the excited‐state intramolecular proton transfer (ESIPT) process of 2‐(2'‐hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. Upon phosphorylated with POCl₃, HPBI shows only an emission peak at 363 nm due to the blockage of ESIPT. However, once selective enzymatic hydrolysis with alkaline phosphatase (ALP) in Tris–HCl buffer occurs, the probe 1 is returned to HPBI and the ESIPT process is switched on, which results in a decrease in the emission band at 363 nm and an increase in a new fluorescence peak around 430 nm. The fluorescence intensity ratio at 430 and 360 nm (I₄₃₀/I₃₆₀) increases linearly with the activity of ALP up to 0.050 U/mL and the detection limit is 0.0013 U/mL. The proposed probe shows excellent specificity toward ALP. Copyright © 2015 John Wiley & Sons, Ltd.     

Fluorescence emission spectroscopy of 1,4-dihydroxyphthalonitrile. A method for determining intracellular pH in cultured cells.

We have developed new methodology for measuring intracellular pH (pHi) in cultured cell monolayers and epithelia by analyzing the emission spectra of the trapped fluorescent pH probe, 1,4-dihydroxyphthalonitrile (1,4-DHPN). This compound is unique since both its acid and base forms possess different fluorescence emission characteristics that can be used to quantitate pHi. The fluorescence difference spectrum between an acid and alkaline solution of 1,4-DHPN has a maximum at 455 nm and a minimum at 512 nm. By determining the ratio of the intensity at these two wavelengths as a function of pH, a calibration curve was constructed. Since the two intensities are determined simultaneously, the measurement is independent of dye concentration, bleaching, and intensity fluctuation of the excitation source. Furthermore, analysis of the emission spectra permitted the detection of light scattering, binding effects, and chemical modification of the probe. A microspectrofluorometer was constructed to analyze low light level emission spectra from intracellular 1,4-DHPN. The instrument consists of a modified Leitz inverted microscope (E. Leitz, Inc., Rockleigh, NJ) with a Ploem illuminator adapted for broadband excitation and objective focusing capability. The emission spectra were collected by focusing the fluorescence from the cell onto the entrance slit of an imaging monochromator, which was scanned by a SIT camera interfaced with a computer. This permitted the acquisition of fluorescence emission spectra extending from 391-588 nm in approximately 33 ms. pHi measured in the cultured toad kidney epithelial cell line, A6, was 7.49 +/- 0.04 (n = 12) with an external pH of 7.6. A6 cells were found to regulate pHi in response to both acute acid and alkali loads and maintained pHi relatively constant over a wide range of external pH values. The technique described in this report overcomes several of the difficulties encountered with other fluorescent pH probes where excitation spectroscopy is required to monitor pH.     

Styryl-BODIPY based red-emitting fluorescent OFF-ON molecular probe for specific detection of cysteine

We have synthesized a styryl boron-dipyrromethene (BODIPY)/2,4-dinitrobenzenesulfonyl (DNBS) dyad based red-emitting molecular probe for specific detection of cysteine among the biological thiols. The probe shows intensive absorption at 556 nm and the probe is non-fluorescent. The DNBS moiety can be cleaved off by thiols, the red emission of the BODIPY fluorophore at 590 nm is switched on, with an emission enhancement of 46-fold. The probe shows good specificity toward cysteine over other biological molecules, such as glutathione and amino acids. The emission of the probe is pH-independent in the physiological pH range. The probe is used for fluorescent imaging of cellular thiols. Theoretical calculations based on density functional theory (DFT) were used to elucidate the fluorescence sensing mechanism of the probe, which indicate a dark excited state (S(1)) for the probe but an emissive excited state (S(1)) for the cleaved product (i.e. the fluorophore).     

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.     

A fluorescent probe study of salmine AI

A fluorescent probe, 1-p-toluidinylnapthalene-8-sulfonate (1,8-TNS), was used to study the nonpolar sites on salmine AI. Fluorescence enhancement resulting from binding between the probe and the protein occurs at a wavelength of maximum emission of 497-500 nm, indicating the existence of moderately nonpolar binding sites on salmine AI.Fluorescence enhancement decreases as the ionic strength of the solvent is increased from 0.002 M to 0.050 M. Fluorescence increases with increasing acidity although this effect is not correlated to the pKa of 1,8-TNS. Positive cooperative binding takes place between 1,8-TNS and salmine AI. Equilibrium dialysis indicates that binding occurs only under conditions resulting in significant fluorescent enhancement. The binding was also studied using thin film dialysis, which is much faster than equilibrium dialysis and avoids the observed changes in probe-protein interaction that occur over long time periods with the latter system.     

Adaptability of myosin V studied by simultaneous detection of position and orientation

We studied the structural dynamics of chicken myosin V by combining the localization power of fluorescent imaging with one nanometer accuracy (FIONA) with the ability to detect angular changes of a fluorescent probe. The myosin V was labeled with bifunctional rhodamine on one of its calmodulin light chains. For every 74 nm translocation, the probe exhibited two reorientational motions, associated with alternating smaller and larger translational steps. Molecules previously identified as stepping alternatively 74-0 nm were found to actually step 64-10 nm. Additional tilting often occurred without full steps, possibly indicating flexibility of the attached myosin heads or probing of their vicinity. Processive myosin V molecules sometimes shifted from the top to the side of actin, possibly to avoid an obstacle. The data indicate marked adaptability of this molecular motor to a nonuniform local environment and provide strong support for a straight-neck model of myosin V in which the lever arm of the leading head is tilted backwards at the prepowerstoke angle.     

A deep-red xanthene-based highly sensitive fluorescent probe for detection of hypochlorite

As an oxidant, deodorant and bleaching agent, the hypochlorous acid (HClO) and hypochlorite (ClO⁻) are widely used in corrosion inhibitors, textile dyes, pharmaceutical intermediates and in our daily lives. However, excess usage or aberrant accumulation of ClO⁻ leads to tissue damage or some diseases and even cancer. Therefore, it is necessary to develop a fluorescent probe that specifically identifies ClO⁻. In this article, we synthesized a deep-red xanthene-based fluorescent probe (XA-CN). The strong electron deficient group dicyano endows the probe XA-CN deep-red fluorescent emission with high solubility, selectivity and sensitivity for ClO⁻ detection. Studies showed that the probe demonstrated turn-off fluorescence (643 nm) at the presence of ClO⁻ in dimethylsulfoxide/phosphate-buffered saline 1:1 (pH 9) solution with a limit of detection of 1.64 μM. Detection mechanism investigation revealed that the electron deficient group -CN and the hydroxyl group was oxidized into aldehyde or carbonyl groups at the presence of ClO⁻, resulting ultraviolet-visible absorption of the probe blue shifted and turned-off fluorescence. Furthermore, XA-CN was successfully used for the detection of ClO⁻ in tap water samples.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。     

Spectrofluorometric measurements of the dispersion state of pyrenedodecanoic acid and its uptake by cultured cells and liposomes

Pyrene dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene is covalently linked, showed a considerable increase in fluorescence when the probe was introduced into a hydrophobic environment. Also, when closely packed in an aggregate, an energy transfer between two adjacent molecules of pyrene occurred, resulting in a shift of the peak of the emission spectrum from 378 nm ('monomeric') to 475 nm ('excimeric'). These two respective properties were utilized for the following: (a) A spectrofluorometric measurement of the critical micellar concentration (CMC) of the pyrene fatty acid, defined as the concentration at which the 475 nm emission peak appeared as a consequence of the aggregation of P12 molecules in aqueous solution to form micelles; the CMC of P12 was found to be in the range of 1 to 2 microM. (b) The penetration of P12, from an aqueous solution or dispersion, into unilamellar phospholipid vesicles was determined by monitoring the increase of the fluorescence at 378 nm. The fluorescence increase was time-dependent and proportional to the respective concentrations of P12 or phospholipid vesicles. Substituting the neutral phosphatidylcholine with the negatively-charged phosphatidylserine vesicles resulted in a slower rate as well as lesser total uptake of P12. (c) The uptake of P12 by cells was accompanied by an increase in the monomeric fluorescence emission intensity. Using cells in suspension, this could be followed continuously in a spectrofluorometer equipped with a recorder. The uptake was found to be time-dependent and proportional to P12 concentration.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.     

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.