Airway surface liquid pH is not acidic in children with cystic fibrosis.

Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans. In order to address this deficiency, ASL pH was measured in vivo in children using a novel luminescent technology integrated with fibre-optic probes. Here we show that ASL pH in children with CF is similar to that of children without CF. Findings were supported by highly controlled direct pH measurements in primary human airway epithelial cell culture models, which also suggest that the potential ASL pH gradient produced by defective apical ion transport is balanced out by paracellular shunting of acid/base. Thus, reduced baseline ASL pH is unlikely to be an important pathobiological factor in early CF lung disease.

Small molecule phosphorescent probes for O2 imaging in 3D tissue models.

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 µM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 µs in air-saturated to 57 µs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Complexes of Ir(III)-octaethylporphyrin with peptides as probes for sensing cellular O2.

Ir(III)-porphyrins are a relatively new group of phosphorescent dyes that have potential for oxygen sensing and labeling of biomolecules. The requirement of two axial ligands for the Ir(III) ion permits simple linkage of biomolecules by a one-step ligand-exchange reaction, for example, using precursor carbonyl chloride complexes and peptides containing histidine residue(s). Using this approach, we produced three complexes of Ir(III)-octaethylporphyrin with cell-penetrating (Ir1 and Ir2) and tumor-targeting (Ir3) peptides and studied their photophysical properties. All of the complexes were stable and possessed bright, long-decay (unquenched lifetimes exceeding 45 µs) phosphorescence at around 650 nm, with moderate sensitivity to oxygen. The Ir1 and Ir2 complexes showed positive staining of a number of mammalian cell types, thus demonstrating localization similar to endoplasmic reticulum and ATP- and temperature-independent intracellular accumulation (direct translocation mechanism). Their low photo- and cytotoxicity allows intracellular oxygen to be probed.

Preparation and Characterization of Chromium(III)-Activated Yttrium Aluminum Borate: A New Thermographic Phosphor for Optical Sensing and Imaging at Ambient Temperatures.

A new thermographic phosphor based on chromium(III)-doped yttrium aluminum borate (YAB) is obtained as single crystals by high temperature flux growth and as a microcrystalline powder via solution combustion synthesis. The phosphor is excitable both in the blue (lambda(max) 422 nm) and in the red part of the spectrum (lambda(max) 600 nm) and shows bright NIR emission. The brightness of the phosphor is comparable to that of a well-known lamp phosphor Mn(IV)-doped magnesium fluorogermanate. At ambient temperatures, the Cr(III)-doped YAB shows high temperature dependence of the luminescence decay time, which approaches 1% per deg. The material shows no decrease in luminescence intensity at higher temperatures. The new phosphor is particularly promising for applications in temperature-compensated optical chemosensors (including those based on NIR-emitting indicators) and in pressure-sensitive paints.

Ultrabright oxygen optodes based on cyclometalated iridium(III) coumarin complexes.

Cyclometalated iridium(III) coumarin complexes represent new types of probes for optical oxygen sensing. In comparison to the most commonly used ruthenium(II) polypyridyl dyes and porphyrin complexes with platinum group metals, they possess much more efficient visible absorption and higher quantum yields, which results in much higher brightnesses. Spectral properties and oxygen sensitivity can be fine-tuned by varying the nature of the coumarin ligand and using respective monomeric or dimeric complexes. When incorporated in a model polystyrene film the probes show optimal dynamics of luminescence decay time for oxygen monitoring in the range from 0% to 100% air saturation. Cross-sensitivity to temperature is significantly lower than for the commonly used ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline oxygen probe. The probes, however, exhibit significantly lower photostability, which restricts their application. If long-term measurements are not required, the probes can be successfully used for reliable monitoring of oxygen concentration. High brightness of the complexes makes them particularly attractive for application in thin films (for monitoring of fast processes) and various types of nano- and microparticles, including magnetic beads. If temperature compensation is not applied, the novel optodes result in the lower errors in determination of oxygen content.

In vivo phosphorescence imaging of pO2 using planar oxygen sensors.

OBJECTIVE: Oxygen-dependent quenching of luminescence of metal porphyrin complexes has been used to image the pO(2) distribution over tumor and normal tissue. METHODS: An experimental setup is described using a platinum(II)-octaethyl-porphyrin immobilized in a polystyrene matrix as transparent planar sensor. RESULTS: Sensitivity over a broad range is high at low pO(2) values (+/- 0.2 mm Hg at 0 mm Hg; +/- 1.5 mm Hg at 160 mm Hg pO(2)). Due to intrinsically referencing via lifetime encoding there was no modification of the sensor response in vivo in the dorsal skinfold chamber model with amelanotic melanoma (A-MEL-3) in awake hamsters when compared to the in vitro calibration. pO(2) measurements over normal tissue (25.8 +/- 5.1 mm Hg) and tumor tissue (9.2 +/- 5.1 mm Hg) were in excellent agreement with previous results obtained in this model using a surface multiwire electrode. CONCLUSIONS: Using the presented method the surface pO(2) distribution can be mapped with a high temporal resolution of approximately 100 ms and a spatial resolution of at least 25 mu m. Moreover, the transparent sensor allows the simultaneous visualization of the underlying microvasculature.

Gas sensing in microplates with optodes: influence of oxygen exchange between sample, air, and plate material.

Microplates with integrated optical oxygen sensors are a new tool to study metabolic rates and enzyme activities. Precise measurements are possible only if oxygen exchange between the sample and the environment is known. In this study we quantify gas exchange in plastic microplates. Dissolved oxygen was detected using either an oxygen-sensitive film fixed at the bottom of each well or a needle-type sensor. The diffusion of oxygen into wells sealed with different foils, paraffin oil, and paraffin wax, respectively, was quantified. Although foil covers showed the lowest oxygen permeability, they include an inevitable gas phase between sample and sealing and are difficult to manage. The use of oil was found to be critical due to the extensive shaking caused by movement of the plates during measurements in microplate readers. Thus, paraffin wax was the choice material because it avoids convection of the sample and is easy to handle. Furthermore, without shaking, significant gradients in pO2 levels within a single well of a polystyrene microplate covered with paraffin oil were detected with the needle-type sensor. Higher pO2 levels were obtained near the surface of the sample as well as near the wall of the well. A significant diffusion of oxygen through the plastic plate material was found using plates based on polystyrene. Thus, the location of a sensor element within the well has an effect on the measured pO2 level. Using a sensor film fixed on the bottom of a well or using a dissolved pO2-sensitive indicator results in pO2 offset and in apparently lower respiration rates or enzyme activities. Oxygen diffusion through a polystyrene microplate was simulated for measurements without convection--that is, for samples without oxygen diffusion through the cover and for unshaken measurements using permeable sealings. This mathematical model allows for calculation of the correct kinetic parameters.