Evaluation of BCECF fluorescence ratio imaging to properly measure gastric intramucosal pH variations in vivo

2007 ◽  
Vol 12 (6) ◽  
pp. 064014 ◽  
Author(s):  
Philippe Rochon ◽  
Mercé Jourdain ◽  
Jacques Mangalaboyi ◽  
François Fourrier ◽  
Sylvie Soulié-Bégu ◽  
...  
Keyword(s):  
Fluorescence Ratio ◽  
Intramucosal Ph ◽  
Gastric Intramucosal Ph ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Ph Variations

scholarly journals Fluorescence Ratio Imaging Microscopy Shows Decreased Access of Vancomycin to Cell Wall Synthetic Sites in Vancomycin-Resistant Staphylococcus aureus

2007 ◽  
Vol 51 (10) ◽  
pp. 3627-3633 ◽  
Author(s):  
Pedro M. Pereira ◽  
Sérgio R. Filipe ◽  
Alexander Tomasz ◽  
Mariana G. Pinho
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Capacity ◽  
Live Cells ◽  
Resistant Bacteria ◽  
Fluorescence Ratio ◽  
Lipid Ii ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging

ABSTRACT A new method of fluorescence ratio imaging microscopy was used to compare the in vivo binding capacity and the access of a fluorescent derivative of vancomycin to the cell wall synthetic sites in isogenic pairs of vancomycin-susceptible and -resistant laboratory mutants and vancomycin-intermediate and -susceptible clinical isolates of Staphylococcus aureus. Live cells of resistant strains were found to bind approximately 1.5 times more antibiotic, but there was no correlation between the increased binding capacity and the MICs of the strains. In both susceptible and resistant bacteria, the subcellular sites of wall synthesis were localized to the division septa, but the rate of diffusion of drug molecules to these sites was reduced in resistant cells. The findings allow a reinterpretation of the mechanism of vancomycin resistance in which the path of vancomycin to its lethal target (lipid II) is considered to be through the division septum and therefore is dependent on the stage of the staphylococcal cell cycle.

Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy: Evidence for a primary proton pump in the plasmalemma of a marine fungus

1990 ◽  
Vol 96 (4) ◽  
pp. 731-736
Author(s):  
JULIA M. DAVIES ◽  
C. BROWNLEE ◽  
D. H. JENNINGS
Keyword(s):  
Intracellular Ph ◽  
Proton Pump ◽  
Marine Organism ◽  
Marine Fungus ◽  
Primary Proton ◽  
Acetoxymethyl Ester ◽  
Fluorescence Ratio Imaging ◽  
Negative Membrane Potential ◽  
Ratio Imaging

The facultative marine fungus, Dendryphiella salina, has the most negative membrane potential yet recorded for a marine organism. The ionic basis for this is thought to be through the action of a primary proton pump, though there exists the possibility of electrogenic pumping of Na+ or Cl−, given the high ambient concentration of these ions. Fluorescence ratio imaging microscopy with the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein (BCECF) has been used to estimate intracellular pH. Hyphae loaded readily with BCECF after incubation with the acetoxymethyl ester (BCECF/AM). Mean resting intracellular pH (pH1) was 7.3, calculated by comparing 490/450 nm fluorescence ratios with in vivo calibration curves obtained by pH equilibration using nigericin. Distinct pH compartments could be observed, corresponding to cytoplasmic and smaller vacuolar compartments. Sodium azide reversibly reduced pH1 by an average of 0.51 of a pH unit, though the response varied between individual hyphae. Inhibiting the plasmalemma ATPase with orthovanadate also reversibly decreased pH|. The results support the presence of a proton pump in the plasmamembrane. The energetic and evolutionary implications are discussed.

Determination of apoplastic Na+ in intact leaves of cotton by in vivo fluorescence ratio-imaging

2002 ◽  
Vol 29 (12) ◽  
pp. 1491 ◽  
Author(s):  
Karl H. Mühling ◽  
André Läuchli
Keyword(s):  
Mature Leaves ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Compatible Osmolytes ◽  
Concentration Changes ◽  
Sodium Binding ◽  
Leaf Apoplast

Salinity may reduce plant growth via Na+-toxicity symptoms in mature leaves after long-term exposure. It has been suggested by other authors that Na+ accumulates in the leaf apoplast and leads to dehydration of leaves, wilting, and finally to death of these leaves. Two methods were employed to determine the Na+ concentration in the leaf apoplast of salt-tolerant cotton plants under salinity. The ratio imaging of sodium-binding benzofuran isophthalate (SBFI) fluorescence was used to detect in vivo concentration changes and gradients of Na+ within the leaf apoplast under salinity stress, and results were compared with the infiltration–centrifugation method. A�significant increase in Na+ concentration was found in the leaf apoplast under salinity (75 mM NaCl), but no further significant increase was determined when NaCl supply was increased from 75 to 150 mM. Both methods revealed that Na+ concentrations remained relatively low, and thus could not be responsible for the decline in yield under salinity. The ratio images showed changes in Na+ concentration and gradients within the leaf apoplast under salt stress, and demonstrated the validity of the method. However, SBFI fluorescence was also influenced by pH, proteins and salt-induced compatible osmolytes.

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