A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

Can we measure terrestrial photosynthesis from space directly, using spectral reflectance and fluorescence?

Attempts to estimate photosynthetic rate or gross primary productivity from remotely sensed absorbed solar radiation depend on knowledge of the light use efficiency (LUE). Early models assumed LUE to be constant, but now most researchers try to adjust it for variations in temperature and moisture stress. However, more exact methods are now required. Hyperspectral remote sensing offers the possibility of sensing the changes in the xanthophyll cycle, which is closely coupled to photosynthesis. Several studies have shown that an index (the photochemical reflectance index) based on the reflectance at 531 nm is strongly correlated with the LUE over hours, days and months. A second hyperspectral approach relies on the remote detection of fluorescence, which is a directly related to the efficiency of photosynthesis. We discuss the state of the art of the two approaches. Both have been demonstrated to be effective, but we specify seven conditions required before the methods can become operational.

The influence of powdery mildew infection on photosynthesis, chlorophyll fluorescence, leaf chlorophyll and carotenoid content of three woody plant species

The effect of powdery mildew development on photosynthesis, chlorophyll fluorescence, leaf chlorophyll and carotenoid concentrations on three woody plants frequently planted in urban environments was studied. Rates of photosynthetic CO2 fixation were rapidly reduced in two of the three genotypes tested prior to visible signs of infection. Effects on chlorophyll fluorescence (Fo, Fv/Fo, Fv/Fm), leaf chlorophyll and carotenoid content were not manifest until >25 per cent of the leaf area was observed to be covered by mycelial growth indicating reduced photo-synthetic rates during the early stages of infection were not due to degradation of the leaf chloroplast structure. Observation of the fluorescence transient (OJIP curves) showed powdery mildew infection impairs photosynthetic electron transport system by reducing the size but not heterogeneity of the plastoquninone pool, effecting both the acceptor and donor side of photosystem II. Impairment of the photosynthetic electron transport system was reflected by reduced values of a performance index used in this investigation as a measure of photochemical events within photosystem II electron transport. In addition interpretation of the fluorescence data indicated powdery mildew infection may impair the photo-protective process that facilitates the dissipation of excess energy within leaf tissue.

Measurement of the salinity and freezing tolerance of Crataegus genotypes using chlorophyll fluorescence

The effect of increasing salinity and freezing stress singly and in combination on a range of chlorophyll fluorescence parameters in foliar tissue of six Crataegus genotypes was examined. In general, increased stress reduced fluorescence values and absorption, trapping and electron transport energy fluxes per leaf reaction center and cross section, with decreased sigmoidicity of OJIP curves as a measure of the plastoquinone pool, reflecting decreased energy fluxes. Based on percentage reduction in a performance index from controls compared to stress-treated values, plants were ranked in order of tolerant > intermediate > sensitive. Use of this PIp ranking criteria enabled the distinguishing of marked differences in foliar salt/freezing hardiness between the Crataegus species used. Interpretation of the photochemical data showed that salinity and freezing affects both the acceptor and donor side of Photosystem II, while OJIP observations provided information regarding structural and functional changes in the leaf photosynthetic apparatus of the test species. It is concluded that chlorophyll fluorescence offers a rapid screening technique for assessing foliar salinity and freezing tolerance of woody perennials

Foliar salt tolerance of Acer genotypes using chlorophyll fluorescence

The effect of increasing salinity on a range of chlorophyll fluorescence parameters in foliar tissue of 30 Acer genotypes was examined. The magnitude of the fluorescence responses differed among genotypes ranging from minor effects to substantial leaf tissue damage. Interpretation of the fluorescence expressions provided an insight into mechanisms of salt damage and resilience among genotypes. Based on reductions in a performance index (PIp) following salinity, genotypes were ranked in order from tolerant to sensitive. Based on this ranking criterion, marked differences in salt tolerance among genotypes were distinguished. It is concluded that chlorophyll fluorescence offers a rapid screening technique for assessing the foliar salinity tolerance of urban trees.

Technical note: Organics-induced fluorescence in Raman studies of sulfuric acid aerosols

Fluorescence is a troublesome side effect in laboratory Raman studies on sulfuric acid solutions and aerosol particles. We performed experiments showing that organic matter induces fluorescence in H2SO4/H2O solutions. The intensity of the fluorescence signal appears to be almost independent of the concentration of the organic substances, but depends strongly on the sulfuric acid concentration. The ubiquity of organic substances in the atmosphere, their relatively high abundance, and the insensitivity of the fluorescence with respect to their concentrations will render most acidic natural aerosols subject to absorption and fluorescence, possibly influencing climate forcing. We show that, while fluorescence may in the future become a valuable tool of aerosol diagnostics, the concurrent absorption is too small to significantly affect the atmosphere's radiative balance.

Detection of nitroaromatic vapours with diketopyrrolopyrrole thin films: exploring the role of structural order and morphology on thin film properties and fluorescence quenching efficiency

Sensitive optical detection of nitroaromatic vapours with diketo-pyrrolopyrrole thin films is reported for the first time and the impact of thin film crystal structure and morphology on fluorescence quenching behaviour demonstrated.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.