Selective detection of luminescence from semiconductor quantum dots by nanosecond time-gated imaging with a colour-masked CCD detector.

Quantum dots are of considerable interest as highly detectable labels with broad absorption, narrow spectral emission and good quantum yields. The luminescence emission has a longer decay time than that of the most common fluorophores, leading to facile rejection of much background emission (such as autofluorescence from biological samples) by means of gated detection. Here, it is shown that a new technique, true-colour nanosecond time-gated luminescence imaging, can be used for selective detection of quantum dot luminescence and should prove valuable for multiplexed detection on the basis of both spectral emission profile and luminescence decay time.

Prospects for applications of lanthanide-based upconverting surfaces to bioassay and detection.

Biological assays to detect binding interactions are often conducted using fluorescence resonance energy transfer (FRET) but this has several disadvantages that markedly reduce the dynamic range of measurements. The very short range of FRET interactions also causes difficulties when large analytes such as viruses or spores are to be detected. Conventional FRET-based assays can in principle be improved using infrared-excited upconverting lanthanide-based energy donors but this does not address the short range of the FRET process. Here we investigate an alternative mode of energy transfer based on evanescent wave coupling from an erbium-doped waveguide to an absorbed fluorophore and characterise the luminescence from the dopant. The upconverted erbium emission is highly structured with well-separated bands in the violet, green and red spectral regions and very little detectable signal between the peaks. The relative intensity of these bands depends on power-density of infrared excitation. Green emission predominates at low power-density and red emission increases more rapidly as power-density increases, with a smaller violet peak also emerging. The temporal response of the upconverting material to pulsed infrared excitation was investigated and was shown to vary markedly with emission wavelength with the red component being particularly sensitive to the duration of the excitation pulse. A surface monolayer of the fluorescent protein R-phycoerythrin was very easily detected on binding to an upconverting waveguide. The potential advantages and limitations of the evanescent wave excitation technique for fluorescence detection are discussed and avenues for further development are considered.

Positron lifetimes in phospholipid dispersions.

Positron lifetimes have been determined in phospholipid dispersions. In fluid phosphatidylcholines, a lifetime of 3.3 ns is found, and a lifetime of 2.8 ns is found for frozen phosphatidylcholines. In dispersions where fluid and frozen phases coexist due to lateral phase separation, an intermediate lifetime is found.

Lytic effects of melittin and delta-haemolysin from Staphylococcus aureus on vesicles of dipalmitoylphosphatidylcholine.

The effects of the lytic peptides, melittin and delta-haemolysin, are compared in vesicles of gel-phase dipalmitoylphosphatidylcholine (DPPC), using calcein as trapped marker. At low concentration, both toxins cause vesicles to lose contents in 5 mM phosphate buffer near neutral pH, with melittin being the more active. As phosphate concentration is increased, the kinetics of melittin-induced leakage change from a slow, sustained loss to a rapid 'burst' of leakage when melittin is present mainly as tetramer in solution, under conditions where it is reported to lose haemolytic activity towards erythrocytes. At low phosphate concentration, the leakage induced by delta-haemolysin is preceded by a lag phase, though fluorescence measurements show that binding of toxin is rapid. At higher phosphate concentration, the toxin binds rapidly to vesicles, but causes no leakage of entrapped calcein. Steady-state fluorescence spectra show no obvious differences in tryptophan emission for delta-haemolysin bound to lipid in high- or low-phosphate buffer. Spin-label fluorescence-quenching studies show that the single tryptophan residue of delta-haemolysin is buried within the lipid bilayer at all phosphate concentrations used. In gel-phase DPPC, delta-haemolysin shows no tendency to cause vesicle aggregation over several hours, as judged by light scattering, though a slow non-linear effect is seen above the lipid phase transition temperature. These effects are contrasted with those of melittin under similar conditions.

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase.

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.

The phase behaviour of dispersions of Bis-Azo PC: photoregulation of bilayer dynamics via lipid photochromism.

A phospholipid, 1,2-bis(4-(n-butyl)phenylazo-4'-phenylbutyroyl)phosphatidylcholine (Bis-Azo PC), has been synthesised and shown to form stable bilayer vesicles. Light-scattering measurements and differential scanning calorimetry show that a dispersion of the lipid has a cooperative phase transition at a similar temperature to that of dipalmitoylphosphatidylcholine, which Bis-Azo PC resembles in overall size. The phase behaviour of Bis-Azo PC has been investigated by fluorescence spectroscopy and using a series of spin-labelled fatty acid probes. Fluorescence measurements using chlorophyll a as probe sense the onset of the cooperative phase transition, but this is not clearly revealed by any of the spin probes tested. Hysteresis in the phase transition is detected both by light scattering measurements and by fluorescence spectroscopy. No transition is observed for a lipid analogue having a palmitic acid chain and a single azo-containing substituent. Bis-Azo PC is reversibly photochromic, isomerising on exposure to ultraviolet light to a photostationary state mixture where cis isomer predominates. Electron microscopy shows that photoisomerisation decreases average vesicle size, and light scattering and calorimetry demonstrate that the cooperative phase transition is abolished. Illumination with visible light establishes a new photostationary state where trans isomer predominates, and the phase transition is restored. The ability to modulate bilayer phase behaviour reversibly has possible application to relaxation studies of bilayer membrane function, and to drug delivery research.

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions.

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystalline-liquid crystalline phase transitions in phospholipid vesicles of dimyristoly and dipalmitoly phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidylcholine show lateral phase separation and in this system the probes parition preferentially into the more 'fluid' phase. In systems that have been reported to contain 'short range order' or 'liquid clustering', such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.

Photobleaching. A novel fluorescence method for diffusion studies in lipid system.

(1) The fluorescent molecular 12(9-anthroyloxy)-stearic acid dimerises on irradiation with light of 366 nm wavelength. (2) The dimer is nonfluorescent and can be reconverted to the parent compound by irradiation at 254 nm. (3) Kinetic analysis suggests that the dimerisation proceeds by a diffusion-limited second order mechanism in many solvents. (4) Anomalously high rates seen in other systems can be attributed to localised high concentration regions (clusters) of the fluorescent molecule. (5) The analysis has been extended to oriented lipid bilayers and the results suggest that below the gel-liquid crystalline transition temperature the 12(9-anthroyloxy)-stearic acid is excluded by the lipid matrix and forms regions of localised high concentration. (6) In fluid lipid the results suggest an isotropic distribution of the probe. Calculated diffusion coefficients correspond to those found by other techniques.

The use of a phospholipid analogue of diphenyl-1,3,5-hexatriene to study melittin-induced fusion of small unilamellar phospholipid vesicles.

A phospholipid analogue incorporating the diphenyl-1,3,5-hexatriene (DPH) chromophore has been synthesized. The compound has been shown to have similar fluorescence properties to DPH itself but, unlike DPH, is unable to exchange freely through solution when incorporated as probe in a subset of phospholipid vesicles of given composition. The non-exchangeability of this probe has been exploited to study the fusion of phospholipid vesicles to form larger structures. The peptide melittin was used to initiate fusion, and it was shown that vesicles which had been induced to fuse by heating in the presence of melittin would not fuse with subsequently added vesicles.

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles.

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4'-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent 'donor' and 1-(4'-carboxyethyl)-6-(4"-nitro)diphenyl-trans-1,3,5-hexatriene as 'acceptor'. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20 degrees C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with less than 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.

Melittin induces fusion of unilamellar phospholipid vesicles.

Melittin, the soluble lipophilic peptide of bee venom, causes fusion of phospholipid vesicles when vesicle suspensions are heated or cooled through their thermal phase transition. Fusion was detected using a new photochemical method (Morgan, C.G., Hudson, B. and Wolber, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 26-30) which monitors lipid mixing. Electron microscopy and gel filtration confirmed that most of the lipid formed large vesicular structures. Fluorescence experiments with a water-soluble, membrane-impermeable complex of terbium (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690-692) demonstrate that these ionic contents are released during fusion. The large structures formed by melittin-induced fusion are impermeable to these ions and are resistant to further fusion. This is in contrast to the behavior observed for the cationic detergent cetyltrimethylammonium bromide (CETAB). The large size of the vesicles formed, the extreme speed of the fusion event and the appearance of electron microscope images of the vesicles prior to fusion suggest that the mechanism of the fusion process includes a preaggregation step.

Light-induced fusion of liposomes with release of trapped marker dye is sensitised by photochromic phospholipid.

Liposomes have been prepared from dipalmitoylphosphatidylcholine containing small amounts of a synthetic photochromic phospholipid, 'Bis-Azo PC'. In the dark, these are stable at room temperature, and contents do not significantly leak over weeks. Photoisomerisation results in immediate release of trapped marker, and in liposome fusion to form larger structures. Fusion has been detected using a fluorescence polarisation assay, and confirmed by electron microscopy. In mixtures, fusion occurs between 'photochromic' liposomes and those of pure lipid. Bis-Azo PC contains two photochromic acyl chains; analogues bearing a single photochromic chain appear to have little effect on bilayer permeability after isomerisation. Photo-induced leakage and liposome fusion suggest possible applications for localised drug delivery as an adjunct to phototherapy. The ability to non-invasively trigger fusion processes should be useful in fundamental studies of membrane interactions. We believe this to be the first report of photo-induced fusion to date.

Photosensitive liposomes as 'cages' for laser-triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release.

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential 'caging' agents for fast solute release. On photolysis, trapped marker dye can be released from gel-phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene-substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.

Fast solute release from photosensitive liposomes: an alternative to 'caged' reagents for use in biological systems.

The kinetics of release of soluble marker trapped in liposomes of gel phase phospholipid containing a photoisomerisable phospholipid analogue have been investigated. Marker release is triggered by UV laser flash photolysis at 355 nm. A markedly temperature-dependent release rate is seen, and above 25 degrees C millisecond release kinetics can be achieved. These results suggest that such liposomes might find application as an alternative to conventional 'caged' reagents for photo-triggered reagent release in biological research.

Characterization of myomodulin-related peptides from the pulmonate snail Helix aspersa.

Three myomodulin-related peptides--pQLSMLRLamide, PMSMLRLamide, and SLGMLRLamide--have been purified and sequenced from extracts of whole snails. The level of immunoreactive myomodulin was shown by HPLC and RIA to be widely distributed among 26 different snail tissues, with the highest levels (higher even than those in the central ganglia) occurring in certain male reproductive organs. Synthetic pQLSMLRLamide modified either the spontaneous rhythmic activity or the resting tone of several isolated muscular organs: the aorta, ventricle, upper gut, epiphallus, flagellum, and spermatheca; but the retractor muscles of the pharynx, penis, and tentacle were unaffected.

Active uptake of drugs into photosensitive liposomes and rapid release on UV photolysis.

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-L-alpha-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4'-n-butylphenyl)azo-4'-(gamma-phenylbutyroyl))-glycero-3- phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30 degrees C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photo-induced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.

Influence of fluorocarbon emulsions on porphyrin-sensitised oxidation of histidine.

The influence of fluorocarbon emulsions on the efficiency of photosensitized oxidation of histidine in solution has been studied, using haematoporphyrin and dihaematoporphyrin derivatives as sensitisers. It is shown that the fluorocarbon emulsions at low concentrations efficiently disaggregate porphyrins, and thereby enhance photosensitised oxidation. The high solubility of oxygen in fluorocarbon emulsions maintains solution oxygen tension, optimising photooxidative damage. It is suggested that fluorocarbon emulsions might find a role in photodynamic therapy, both as carriers for sensitising dyes, and also to maintain tissue oxygenation in hypoxic regions of solid tumours.

Dye-sensitized destabilization of liposomes bearing photooxidizable lipid head groups.

Liposomes were prepared from mixtures of dipalmitoyl-L-alpha-phosphatidylcholine and up to 40% mol:mol of N-stearoyl-L-histidine (NSH) in the presence of the hydrophobic sensitizer DHE. In the dark such liposomes are stable and retain entrapped salts. On photolysis with visible light, liposomes leak trapped ions at NSH concentrations greater than 10% mol:mol. Up to 15% mol:mol NSH concentration leakage is seen only during the illumination period, whereas at higher concentration the liposomes continue to leak contents after illumination and fuse to form larger structures. Photolysis of the liposomes is accompanied by oxygen uptake in proportion to the NSH concentration within the bilayer. Photocontrol of liposome permeability through oxidation of membrane additives such as NSH offers a potential means for controlled drug delivery and might be useful as an adjunct to photodynamic therapy.

Liposome fusion and lipid exchange on ultraviolet irradiation of liposomes containing a photochromic phospholipid.

A photochromic phospholipid, 1,2-bis[4-4(4-n-butylphenylazo) phenylbutyroyl] phosphatidylcholine (Bis-Azo PC) has been incorporated into liposomes of gel- and liquid-crystalline- phase phospholipids. Liposomes of gel-phase phospholipid are stable in the presence of the trans photostationary state Bis-Azo PC and can encapsulate fluorescent marker dye. On photoisomerization to the cis photostationary state, trapped marker is rapidly released. Liposomes containing Bis-Azo PC can rapidly fuse together after UV isomerization, this process continuing in the dark. Exposure to white light causes reversion of Bis-Azo Pc to the trans form and halts dye leakage and vesicle fusion. Both unilamellar and multilamellar liposomes are able to fuse together on UV exposure. On UV photolysis, liposomes containing Bis-Azo PC do not fuse with a large excess of unlabeled liposomes, but transfer of Bis-Azo PC can be demonstrated spectrophotometrically. Vesicles of pure gel-phase lipid containing trapped marker dye but initially no Bis-Azo PC become leaky as a result of this lipid transfer. Liposomes composed of liquid-crystalline-phase phosphatidylcholine- containing Bis-Azo PC neither leak trapped marker no fuse together on photolysis, nor do liquid-crystalline-phase liposomes fuse with gel-phase liposomes under these conditions. These results are discussed together with some possible applications of liposome photodestabilization.

Validity, reliability, and calibration of the Tritrac accelerometer as a measure of physical activity.

PURPOSE: The purposes of this study were to assess the validity and reliability of the Tritrac R3D accelerometer during treadmill walking and running and then to calibrate the instrument. METHODS: The Tritrac was assessed on 60 young adults (23.4 +/- 2.9 yr) during treadmill walking and running at 3.2, 6.4, and 9.7 km x h(-1). The calibration was carried out by identifying ranges of Tritrac raw data (vector magnitude) values corresponding to light (2-3.9 MET), moderate (4-7 MET), and vigorous (>7 MET) physical activity. Energy expenditure (EE), measured by indirect calorimetry, served as the criterion measure. RESULTS: Interinstrument intraclass reliability coefficients for Tritracs worn on the right and left hip ranged from 0.73-0.87, while intersession coefficients demonstrated high reliability for all speeds (R = 0.87-0.92). Paired t-tests comparing mean accelerometer counts at 6.4 km x h(-1), 0% grade (2647 +/- 456), and 6.4 km x h(-1), 5% grade (2635 +/- 435) demonstrated no significant difference (P > 0.05). Mean differences between EE measured by indirect calorimetry and that estimated by the Tritrac ranged from 0.0082 kcal x kg(-1) x min(-1) at 3.2 km x h(-1) to 0.0320 kcal x kg(-1) x min(-1) at 9.7 km x h(-1), with the Tritrac consistently overestimating EE during horizontal treadmill walking. The relationship between vector magnitude and EE across all speeds was highly linear (R2 = 0.90, SEE = 0.014 kcal x kg(-1) x min(-1)), with little overlap between light, moderate, and vigorous categories. The mean vector magnitudes at 2, 4, and 7 MET were 650, 1772, and 3455, respectively. CONCLUSIONS: These data indicate that the Tritrac is highly reliable from day to day and is sensitive to changes in speed but not grade. Furthermore, the Tritrac accurately distinguishes various intensities of walking and jogging on level ground. With limitations, these cut-points can be used to categorize light, moderate, and vigorous physical activity and to estimate EE.