Combined imaging of bacteria and oxygen in biofilms

Transparent sensors for microscopic O(2) imaging were developed by spin coating an ultrathin (<1- to 2-microm) layer of a luminescent O(2) indicator onto coverslips. The sensors showed (i) an ideal Stern-Volmer quenching behavior of the luminescence lifetime towards O(2) levels, (ii) homogeneous measuring characteristics over the sensor surface, and (iii) a linear decline of luminescence lifetime with increasing temperature. When a batch of such coverslip sensors has been characterized, their use is thus essentially calibration free at a known temperature. The sensors are easy to use in flow chambers and other growth devices used in microbiology. We present the first application for combined imaging of O(2) and bacteria in a biofilm flow chamber mounted on a microscope equipped with a spinning-disk confocal unit and a luminescence lifetime camera system.     

Ultrabright planar optodes for luminescence life-time based microscopic imaging of O₂ dynamics in biofilms

New transparent optodes for life-time based microscopic imaging of O₂ were developed by spin-coating a μm-thin layer of a highly luminescent cyclometalated iridium(III) coumarin complex in polystyrene onto glass cover slips. Compared to similar thin-film O₂ optodes based on a ruthenium(II) polypyridyl complex or a platinum(II) porphyrin, the new planar sensors have i) higher brightness allowing for much shorter exposure times and thus higher time resolution, ii) more homogeneous and smaller pixel to pixel variation over the sensor area resulting in less noisy O₂ images, and iii) a lower temperature dependency simplifying calibration procedures. We used the new optodes for microscopic imaging of the spatio-temporal O₂ dynamics at the base of heterotrophic biofilms in combination with confocal imaging of bacterial biomass and biofilm structure. This allowed us to directly link biomass distribution to O₂ distribution under both steady state and non-steady state conditions. We demonstrate that the O₂ dynamics in biofilms is governed by a complex interaction between biomass distribution, mass transfer and flow that cannot be directly inferred from structural information on biomass distribution alone.     

Complexation, luminescence and energy transfer in the Ce(III)– 2,2′-bipyridine complexes

Fluorescence characteristics and energy transfer to a cerium complex with 2,2′-bipyridine (bpy) in methanol are described. Stoichiometries and the stability constant of the Ce(III)–bpy complex in methanol were determined by use of the molar ratio method. A fluorescence lifetime and a quantum yield have been measured for the 2:1 complex. Decay times and time-resolved (T---S) emission spectra for Ce(III)–bpy and CeCl₃ were measured in methanol at room temperature. An energy transfer rate constant was determined from the luminescence lifetime and the quantum yield. The mechanism of energy transfer from the lowest excited singlet S₁ to the 5d level of the Ce(III) in the Ce(III)–bpy complex system is discussed in some detail.     

An Improved Ras Sensor for Highly Sensitive and Quantitative FRET-FLIM Imaging

Ras is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors'' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (K_d∼1.7 µM) and FRas2-M (K_d∼0.5 µM). We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons.     

Detection of Active Oxygen Species in Biological Systems

1. Cypridina luciferin analogues, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLD) and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one(CLA), react with O₂ ^– or ¹O₂ to emit light in visible region. Such chemiluminescences were used for the detection of O₂ ^– or ¹O₂ in activated leukocyte systems and myeloperoxidase (granulocyte-extract) + Br^– + H₂O₂ systems in vitro.2. The mechanism of MCLA (CLA)-dependent luminescence is described in detail. Superoxide generated from sinusoidal cells in acute ethanol intoxication of rats was detected by MCLA-dependent luminescence from the surface of perfused rat liver (organ luminescence).3. Furthermore, with alive animals, O₂ ^– generated in the lung of rats with necrotized pancreatitis and that in the stomach of rats after ischemia/reperfusion were detected by their organ luminescences.     

Luminescent sensing and imaging of oxygen: Fierce competition to the Clark electrode

Luminescence‐based sensing schemes for oxygen have experienced a fast growth and are in the process of replacing the Clark electrode in many fields. Unlike electrodes, sensing is not limited to point measurements via fiber optic microsensors, but includes additional features such as planar sensing, imaging, and intracellular assays using nanosized sensor particles. In this essay, I review and discuss the essentials of (i) common solid‐state sensor approaches based on the use of luminescent indicator dyes and host polymers; (ii) fiber optic and planar sensing schemes; (iii) nanoparticle‐based intracellular sensing; and (iv) common spectroscopies. Optical sensors are also capable of multiple simultaneous sensing (such as O₂ and temperature). Sensors for O₂ are produced nowadays in large quantities in industry. Fields of application include sensing of O₂ in plant and animal physiology, in clinical chemistry, in marine sciences, in the chemical industry and in process biotechnology.     

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made  possible the measuring of cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H₂O₂ signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor are coated with a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H₂O₂. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H₂O₂ on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the in vivo studies are instead based on the reduction of H₂O₂ on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H₂O₂ and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H₂O₂ in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.     

Photophysical properties of Eu(SiW11O39)2 and Eu(BW11O39)2

The photophysical properties (absorption, emission, and excitation spectra; luminescence quantum yields; luminescence decay lifetimes ) of K₁₃[Eu(SiW₁₁O₃₉)₂] and K₁₅[Eu(BW₁₁O₃₉)₂] in aqueous solution and in the solid state are reported. Both complexes exhibit broad and very intense O → W charge transfer bands in the U.V. region and weak and narrow f → f Eu₃₊ bands in the visible. At 77 K the luminescence emission of both complexes, which consists of ⁵D_O → ⁷F_J bands split by the local crystal field, can be pumped very efficiently via both the O → W CT and the f → f Eu³⁺ levels, whereas at 298 K only pumping via the f → f Eu³⁺ is efficient. The values of the luminescence decay lifetimes in H₂O and D₂O solution are quite similar, showing that no water molecule is coordinated to the central Eu₃₊ ion. The high resolution emission spectra are discussed in an attempt to define the coordination symmetry of Eu₃₊.     

Luminescence of the near‐ultraviolet blue‐emitting Ba9Al2Si6O24:Ce3+ phosphor

A near‐ultraviolet (NUV) blue‐emitting phosphor Ba₉Al₂Si₆O₂₄:Ce³⁺ (BAS:Ce³⁺) was synthesized using a high‐temperature solid‐state reaction. BAS:Ce³⁺ had an excitation band peak at about 328 nm and showed a blue emission band. The NUV‐blue emission band had a peak at about 386 nm with a band width of about 60 nm, attributed to the 5d–4f transition of Ce³⁺. Fluorescent decay showed an exponential model with a lifetime of 27.2 nsec. At 150°C, the luminescence intensity decreased to 68.7% compared with the intensity at room temperature.     

IMAGING OF OXYGEN DYNAMICS WITHIN THE ENDOLITHIC ALGAL COMMUNITY OF THE MASSIVE CORAL PORITES LOBATA1

We used transparent planar oxygen optodes and a luminescence lifetime imaging system to map (at a pixel resolution of <200 μm) the two‐dimensional distribution of O₂ within the skeleton of a Porites lobata colony. The O₂ distribution was closely correlated to the distribution of the predominant endolithic microalga, Ostreobium quekettii Bornet et Flahault that formed a distinct green band inside the skeleton. Oxygen production followed the outline of the Ostreobium band, and photosynthetic O₂ production was detected at only 0.2 μmol photons m^?2 · s^?1, while saturation occurred at ～37 μmol photons m^?2 · s^?1. Oxygen levels varied from ～60% to 0% air saturation in the illuminated section of the coral skeleton in comparison to the darkened section. The O₂ production within the Ostreobium band was lower in the region below the upward facing surface of the coral and elevated on the sides. Oxygen consumption in darkness was also greatest within the Ostreobium zone, as well as in the white skeleton zone immediately below the corallites. The rate of O₂ depletion was not constant within zones and between zones, showing pronounced heterogeneity in endolithic respiration. When the coral was placed in darkness after a period of illumination, O₂ levels declined by 50% within 20 min and approached steady‐state after 40–50 min in darkness. Our study demonstrates the use of an important new tool in endolith photobiology and presents the first data of spatially resolved O₂ concentration and its correlation to the physical structures and specific zones responsible for O₂ production and consumption within the coral skeleton.     

Luminescent inorganic mixed borate phosphors materials for lighting

The need of new materials with desirable optical properties has become important in recent years. In particular, a need has emerged for compounds having better luminescence properties in various practical applications. The introduction of rare earth ions as activators improves the luminescence properties of the compounds considerably. Boron is one of the most abundant elements in nature. It readily combines with almost all other elements. In combination with oxygen; it forms borates comprised of various anions such as BO₃³⁻, B₂O₅⁴⁻, etc. Apart from these simple borates, several complex compositions exist involving mixed anions as well as double metal borates, due to the three-fold, or four-fold coordination of borate atoms. Borates intrinsically possesses characteristics that are advantageous for optical materials, which include a wide transparency range, large electronic band gap, good thermal and chemical stability, low preparative temperature, optical stability with good nonlinear characteristics, and an exceptionally high optical damage threshold. The unique crystal structure of borates determines their enhanced ultraviolet light transparency, good nonlinearity, and relatively high resistance against laser-induced damage. Some of these complex borates have interesting luminescence properties that are covered in this review. These include double borates containing rare earths RM₃(BO₃)₄, pentaborates LaMgB₅O₁₀, M₃R₂(BO₃)₄, where M is an alkaline earth, mixed anion borates such as aluminoborate SrAl₂B₂O₇, silicate-borates such as pekovite, SrB₂Si₂O₈, haloborates, M₂B₅O₉X, where M is an alkaline earth and X is a halogen, phosphate borates, and MBPO₅, where M is an alkaline earth. Phosphors based on these compositions find use in various applications such as fluorescence lamps, colour TVs, plasma display panels, high-intensity discharge lamps based on xenon, optically pumped solid-state lasers, eye-safe lasers, and X-ray imaging.     

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H₂O₂) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H₂O₂ sensor that allows H₂O₂ monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H₂O₂ sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔC_R to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H₂O₂ produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H₂O₂ levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H₂O₂ levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H₂O₂ biology in plant cells.

The establishment of a hypersensitive H₂O₂ sensor in six major compartments of the Chlamydomonas cell reveals steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments.     

Xylem sap flow as a major pathway for oxygen supply to the sapwood of birch (Betula pubescens Ehr.)

The role of xylem sap flow as an aqueous pathway for oxygen supply to the wood parenchyma of Betula pubescens saplings was investigated. Using micro‐optode sensors the oxygen status of the sapwood was quantified in relation to mass flow of xylem sap. Sap flow was gradually reduced by an increasing oxygen depletion in the root space. The effect of sap flow on radial O₂ transport between stem and atmosphere was assessed by a stoichiometrical approach between respiratory CO₂ production and O₂ consumption. Restriction of sap flow set in 36.5 h after the onset of O₂ depletion, and was complete after 71 h. Interruption of sap flow drastically increased the O₂ deficit in the sapwood to 70%. Sap flow contributed about 60% to the total oxygen supply to the sapwood. Diurnal O₂ flow rates varied between 3 and 6.3 nmol O₂ m^?2 leaf area (LA) s^?1 during night‐ and daytime, respectively. Maximum O₂ flow rates of 20 nmol O₂ m^?2 LA s^?1 were reached at highest sap flow rates of 5.7 mmol H₂O m^?2 LA s^?1. Sap flow not only affected the oxygen status of the sapwood but also had an effect on radial O₂ transport between stem and atmosphere.     

Near Infrared Imaging of EGFR of Oral Squamous Cell Carcinoma in Mice Administered Arsenic Trioxide

Background

The effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR) expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was studied in mice.

Material and Methods

A431 OSCC in vitro were exposed to 0 µM, 0.5 µM, 2.5 µM, or 5 µM of As₂O₃ for 0 h, 24 h, 48 h and 72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related signal decline with As₂O₃ treatment. Next, mice with pharynx-implanted A431 cells received As₂O₃ i.p. every 48 h at 0.0, 0.5, 2.5, or 5 mg/kg/day (n = 6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.

Results

In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p<0.05) with increasing As₂O₃ dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p<0.05) in As₂O₃ treated groups versus controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was reduced by As₂O₃ treatment in a dose responsive pattern.

Conclusion

This study demonstrates for the first time that OSCC can be interrogated in vivo by NIR molecular imaging of the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As₂O₃ treatment.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.     

Chilling-enhanced photooxidation : evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants

Chilling temperatures (5°C) and high irradiance (1000 microeinsteins per square meter per second) were used to induce photooxidation in detached leaves of cucumber (Cucumis sativus L.), a chilling-sensitive plant. Chlorophyll a, chlorophyll b, β carotene, and three xanthophylls were degraded in a light-dependent fashion at essentially the same rate. Lipid peroxidation (measured as ethane evolution) showed an O₂ dependency. The levels of three endogenous antioxidants, ascorbate, reduced glutathione, and α tocopherol, all showed an irradiance-dependent decline. α-Tocopherol was the first antioxidant affected and appeared to be the only antioxidant that could be implicated in long-term protection of the photosynthetic pigments. Results from the application of antioxidants having relative selectivity for ¹O₂, O₂⁻, or OH indicated that both ¹O₂ and O₂⁻ were involved in the chilling- and light-induced lipid peroxidation which accompanied photooxidation. Application of D₂O (which enhances the lifetime of ¹O₂) corroborated these results. Chilling under high light produced no evidence of photooxidative damage in detached leaves of chilling-resistant pea (Pisum sativum L.). Our results suggest a fundamental difference in the ability of pea to reduce the destructive effects of free-radical and ¹O₂ production in chloroplasts during chilling in high light.     

Comparison of techniques for the measurement of skin temperature during exercise in a hot,humid environment

Exercising or working in a hot, humid environment can results in the onset of heat-related illness when an individual''s temperature is not carefully monitored. The purpose of the present study was to compare three techniques (data loggers, thermal imaging, and wired electrodes) for the measurement of peripheral (bicep) and central (abdominal) skin temperature. Young men and women (N = 30) were recruited to complete the present study. The three skin temperature measurements were made at 0 and every 10-min during 40-min (60% VO₂max) of cycling in a hot (39±2°C), humid (45±5% RH) environment. Data was statistically analyzed using the Bland-Altman method and correlation analysis. For abdominal skin temperature, the Bland-Altman limits of agreement indicated that data loggers (1.5) were a better index of wired than was thermal imaging (3.5), For the bicep skin temperature the limits of agreement was similar between data loggers (1.9) and thermal (1.9), suggesting the both were suitable measurements. We also found that when skin temperature exceeded 35°C, we observed progressively better prediction between data loggers, thermal imaging, and wired skin sensors. This report describes the potential for the use of data loggers and thermal imaging to be used as alternative measures of skin temperature in exercising, human subjects.     

THE OXYGEN CONSUMPTION OF LUMINOUS BACTERIA

Oxygen consumption of luminous bacteria determined by the Thunberg micro respirometer and by the time which elapses before the luminescence of an emulsion of luminous bacteria in sea water begins to dim, when over 99 per cent of the dissolved oxygen has been consumed, agree exactly. Average values for oxygen consumption at an average temperature of 21.5°C. are 4.26 x 10^–11 mg. O₂ per bacterium; 2.5 x 10⁴ mg. per kilo and 5.6 mg. O₂ per sq. m. of bacterial surface. The only correct comparison of the oxygen consumption of different organisms or tissues is in terms of oxygen used per unit weight with a sufficient oxygen tension so that oxygen consumption is independent of oxygen tension. Measurement of the oxygen concentration which just allows full luminescence, compared with a calculation of the oxygen concentration at the surface of a bacterial cell just necessary to allow the observed respiration throughout all parts of the cell, indicates that oxygen must diffuse into the bacterium much more slowly than through gelatin or connective tissue but not as slowly as through chitin.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.