Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.     

Fluorescence modeling in a multicomponent system

An extensive fluorescence database for binary tyrosinetryptophan mixtures utilizing 280 nm excitation was collected. The database spanned three orders of magnitude (10(-6)M-10(-3)M) and covered all compositions within this range. A generalized model for describing the multicomponent fluorescence signals as a function of emission wavelength, excitation wavelength, and sample composition was derived. A geometric integral that contained all the geometric factors affecting fluorescence was introduced; thus the model was applicable to various configurations, including the three used in this study: an NADH probe, a backscatter laser-induced fluorescence setup, and a commercial spectroflurometer. A correction factor was proposed that allowed linearization of the fluorescence signals with respect to fluorophore concentrations. The effect of the water Raman on fluorescence spectra was also modeled. The model contains only two wavelength-dependent parameters for each of the components present in a sample, one specifying absorption of the excitation energy and the other specifying the species' fluorescence tendency. These wavelength-dependent parameters were correlated with polynomials. The average prediction error at each wavelength was 10-20%, a major portion of which was attributed to experimental uncertainties.     

Potential charge transfer probe induced conformational changes of model plasma protein human serum albumin: Spectroscopic, molecular docking, and molecular dynamics simulation study

The nature of binding of specially designed charge transfer (CT) fluorophore at the hydrophobic protein interior of human serum albumin (HSA) has been explored by massive blue-shift (82 nm) of the polarity sensitive probe emission accompanying increase in emission intensity, fluorescence anisotropy, red edge excitation shift, and average fluorescence lifetimes. Thermal unfolding of the intramolecular CT probe bound HSA produces almost opposite spectral changes. The spectral responses of the molecule reveal that it can be used as an extrinsic fluorescent reporter for similar biological systems. Circular dichrosim spectra, molecular docking, and molecular dynamics simulation studies scrutinize this binding process and stability of the protein probe complex more closely.     

Use of fluorometry for monitoring and control of a bioreactor

A new fluorescent bioreactor monitoring probe-multiple excitation fluorometric system (MEFS)-has been developed. This probe was compared to the commercially available BioChem Technology FluroMeasure system (NADH probe). In this task the fluorescence behavior of three model fermentation systems, ethanol fermentation by Candida utilis, phenol fermentation by Pseudomonas putida, and glucose fermentation by Saccharomyces cerevisiae, were examined. The results indicated that the fluorescence intensity and behavior of various cellular fluorophors vary significantly between the different fermentation systems. Monitoring a fermentation process using only NAD(P)H fluorescence provided limited information. The NAD(P)H fluorescence was found not to be the best fluorescence signal for monitoring cell concentrations. The best way of monitoring a bioreactor by fluorometry may be to monitor several fluorophors in the whole culture broth simultaneously and to relate these fluorescence signals to various biological parameters.     

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).     

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.     

Wavelength-shifting molecular beacons

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.     

Physical and photophysical characterization of a BODIPY phosphatidylcholine as a membrane probe

Lipids containing the dimethyl BODIPY fluorophore are used in cell biology because their fluorescence properties change with fluorophore concentration (C.-S. Chen, O. C. Martin, and R. E. Pagano. 1997. Biophys J. 72:37-50). The miscibility and steady-state fluorescence behavior of one such lipid, 1-palmitoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (PBPC), have been characterized in mixtures with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). PBPC packs similarly to phosphatidylcholines having a cis-unsaturated acyl chain and mixes nearly ideally with SOPC, apparently without fluorophore-fluorophore aggregation. Increasing PBPC mole fraction from 0.0 to 1.0 in SOPC membranes changes the emission characteristics of the probe in a continuous manner. Analysis of these changes shows that emission from the excited dimethyl BODIPY monomer self quenches with a critical radius of 25.9 A. Fluorophores sufficiently close (< or =13.7 A) at the time of excitation can form an excited dimer, emission from which depends strongly on total lipid packing density. Overall, the data show that PBPC is a reasonable physical substitute for other phosphatidylcholines in fluid membranes. Knowledge of PBPC fluorescence in lipid monolayers has been exploited to determine the two-dimensional concentration of SOPC in unilamellar, bilayer membranes.     

Simple PbII fluorescent probe based on PbII-catalyzed hydrolysis of phosphodiester

A new fluorescent probe for PbII, p-nitrophenyl 3H-phenoxazin-3-one-7-yl phosphoric acid (NPPA), was designed and synthesized by linking resorufin (serving as a fluorophore and electron acceptor) to p-nitrophenol (serving as a fluorescence quencher and electron donor) through phosphodiester bonds. When NPPA was irradiated with light, intramolecular fluorescence self-quenching took place because of the photoinduced electron transfer from the donor to the acceptor. However, upon the addition of PbII, the phosphate ester bonds in the probe were cleaved and the fluorophore was released, accompanying the retrievement of fluorescence.     

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.     

Stem-loop probe with universal reporter for sensing unlabeled nucleic acids

In this article, we present the design principles and application of a motif composed of a stem-loop probe (SP) hybridized to a fluorescently labeled universal reporter (UR) for sensing unlabeled nucleic acids. At room temperature, SP-UR is in the hairpin-closed form in which the fluorophore of UR is in proximity to the G bases of the hairpin, where consequently the fluorescent emission is quenched significantly. On hybridization with target, SP-UR is trapped in the hairpin-opened configuration in which the fluorophore and the G quenchers are apart. This turns off quenching, increases emission intensity, and signals the presence of target. Compared with the common approach that employs an oligonucleotide probe with a covalently linked fluorophore, the use of a fluorescently labeled universal reporter strand hybridized to an unlabeled stem-loop probe provides a more efficient approach to the fabrication of nucleic acid sensors and microarrays potentially useful for real-time analysis.     

肽链及蛋白质N-末端羰酰荧光衍生物的形成

短肽及胰岛素的N-末端经Dixpn转氨后具有荧光发色性质,其激发与发射波长随肽链氨基酸残基的种类和数量的不同而有差异,其范围大约为Ex 312—333nm;Em 398—408nm;量子产率为0.020—0.03.荧光发色团的基本化学结构可能是N-末端α-羰酰基及其相连的酰胺基,并且N-末端第二及第三位氨基酸残基对其荧光性质有影响.在碱性溶液中(pH>9.0)它的荧光强度降低,但在NaCl溶液中随盐浓度的增加而增强.在不同浓度的CuHCl溶液中,羰酰三肽的荧光强度变化随其氨基酸残基的种类和构型的不同而有差异.以上结果提示;α-羰酰荧光衍生物可能作为蛋白及肽N-末端的荧光探剂,可能成为研究蛋白和肽结构与功能的一种手段.     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near-infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and the highly efficient production of signal. While the fluorescence properties of green fluorophores are well-known in vitro, less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed, and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.     

Multicolour flow cytometry analyses and autofluorescence in chlorophytes: lessons from programmed cell death studies in Chlamydomonas reinhardtii

Flow cytometry is a valuable tool in phycological studies. However, endogenous cellular compounds like nicotinamide adenine dinucleotide and chlorophyll a and b autofluoresce, potentially interfering with fluorescent markers. Furthermore, autofluorescent properties are not uniform across algae, nor are their effects consistent in different cytometers. The choice of instrument and fluorescent marker, therefore, requires careful consideration. We investigated the suitability of fluorescent markers by using standard four-colour and advanced multicolour flow cytometers in relation to the effects of autofluorescence over ranges of parameters including fluorophore excitation and emission spectra, band-pass filter configurations, voltage gains and the effects of growth in the light and dark. The unicellular chlorophyte and model organism, Chlamydomonas reinhardtii, was used and findings were correlated with investigations of programmed cell death. As previously found C. reinhardtii autofluoresces in the red, far-red and infrared spectra. This is independent of laser excitation wavelength, and autofluorescence emits and spills over into detection channels of both four-colour and multicolour instruments. Band-pass filter configurations capturing longer wavelength emissions or fluorophores excited or emitted in these longer wavelengths are generally unsuitable. Furthermore, neither dark nor light incubation impacted the autofluorescent signals. Consideration of these algal autofluorescent properties and their spillover effects is required to avoid erroneous results. Recommendations for the use of a range of fluorophores in programmed cell death and other studies in C. reinhardtii using four-colour and multicolour instruments are made.     

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.     

Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences

2-Aminopurine (2-AP), a fluorescent analog of adenine, has been widely used as a probe for local DNA conformation, since excitation and emission characteristics and fluoresence lifetimes of 2-AP vary in a sequence-dependent manner within DNA. Using steady-state and time-resolved fluorescence techniques, we report that 2-AP appears to be unusually stacked in the internal positions of ATAT and TATA in duplex DNA. The excitation wavelength maxima for 2-AP within these contexts were red shifted, indicating reduced solvent exposure for the fluorophore. Furthermore, in these contexts, 2-AP fluorescence was resistant to acrylamide-dependent collisional quenching, suggesting that the fluorophore is protected by its stacked position within the duplex. This conclusion was further reinforced by the presence of a secondary peak at 275 nm in the fluorescence excitation spectra that is indicative of efficient excitation energy transfer from nearby non-fluorescent DNA bases. Fluorescence anisotropy decay and internal angular ‘wobbling’ motion measurements of 2-AP within these alternating AT contexts were also consistent with the fluorophore being highly constrained and immobile within the base stack. When these fluorescence characteristics are compared with those of 2-AP within other duplex DNA sequence contexts, they are unique.     

GFP-tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber

Myosin is the molecular motor in muscle-binding actin and executing a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. A green fluorescent protein (GFP)-tagged human cardiac myosin regulatory light chain (HCRLC) was constructed to study in situ lever arm orientation one molecule at a time by polarized fluorescence emitted from the GFP probe. The recombinant protein physically and functionally replaced the native RLC on myosin lever arms in the thick filaments of permeabilized skeletal muscle fibers. Detecting single molecules in fibers where myosin concentration reaches 300 microM is accomplished using total internal reflection fluorescence microscopy. With total internal reflection fluorescence, evanescent field excitation, supercritical angle fluorescence detection, and CCD detector pixel size limits detection volume to just a few attoliters. Data analysis manages both the perturbing effect of the TIR interface on probe emission and the effect of high numerical aperture collection of light. The natural myosin concentration gradient in a muscle fiber allows observation of fluorescence polarization from C-term GFP-tagged HCRLC exchanged myosin from regions in the thick filament containing low and high myosin concentrations. In rigor, cross-bridges at low concentration at the end of the thick filament maintain GFP dipole moments at two distinct polar angles relative to the fiber symmetry axis. The lower angle, where the dipole is nearly parallel to fiber axis, is more highly populated than the alternative, larger angle. Cross-bridges at higher concentration in the center of the thick filament are oriented in a homogeneous band at approximately 45 degrees to the fiber axis. The data suggests molecular crowding impacts myosin conformation, implying mutual interactions between cross-bridges alter how the muscle generates force. The GFP-tagged RLC is a novel probe to assess single-lever-arm orientation characteristics in situ.     

Multiple wavelength fluorescence enhancement on glass substrates for biochip and cell analyses

In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve the sensitivity of the fluorescence detection of both common fluorophores Cy3 and Cy5 in DNA microarrays and fluorescent cell analyses, we have designed a multi layer mirror with alternative thin layers of SiO2 and HfO2. This mirror (MOTL) prevents fluorescence absorption, allows the simultaneous enhancement of the fluorescence signals and increases the dynamic range of the slides. Using MOTL slides, Cy3 and Cy5 intensities are enhanced by 5-8-fold, consequently, the fluorescence analysis becomes easier and should allow the detection of low copy number genes or weakly fluorescent cells. With the same approach, other multiple optical thin layer slides could be designed for other series of fluorophores, extending the field of their applications.     

Single photon radioluminescence. I. Theory and spectroscopic properties.

The excitation of a fluorescent molecule by a beta-decay electron (radioluminescence) depends upon the electron energy, the distance between radioactive 'donor' and fluorescent 'acceptor', and the excitation characteristics and solvent environment of the fluorophore. The theory for calculation of single photon radioluminescence (SPR) signals is developed here; in the accompanying paper, measurement methods and biological applications are presented. To calculate the three-dimensional spatial profile for electron energy deposition in an aqueous environment, a Monte Carlo calculation was performed incorporating theories of electron energy distributions, energy loss due to interactions with matter, and deflections in electron motion due to collisions. For low energy beta emitters, 50% of energy deposition occurs within 0.63 micron (3H, 18.5 keV), 22 microns (14C, 156 keV), 25 microns (35S, 167 keV), and 260 microns (36Cl, 712 keV) of the radioisotope. In close proximity to the beta emitter (100 nm, 3H; 10 microns, 14C) the probability for fluorophore excitation is approximately proportional to the inverse square of the distance between the beta emitter and fluorophore. To investigate the other factors that determine the probability for fluorophore excitation, SPR measurements were carried out in solutions containing 3H and a series of fluorophores in different solvents. In water, the probability of fluorescence excitation was nearly proportional to the integrated absorbance over a > 1,000-fold variation in absorbances. The probability of fluorescence excitation was enhanced up to 2,600-fold when the fluorophore was in a "scintillant" aromatic or hydrocarbon solvent. SPR emission spectra were similar to fluorescence emission spectra obtained with photon excitation. The single photon signal due to Bremsstrahlung increased with wavelength in agreement with theory. The distance dependence for the SPR signal predicted by the model was in good agreement with measurements in which a 14C donor was separated by known thicknesses of water from a fluorescently-coated coverglass. Quantitative predictions for radioluminescence signal as a function of donor-acceptor distance were developed for specific radioisotope-fluorophore geometries in biological samples.