Simultaneous velocity and pressure measurements using luminescent microspheres.

Using the technique of modified rapid lifetime determination, pressure-sensitive microspheres, known as PrSBeads, were used to make quantitative oxygen measurements over two-dimensional areas within gaseous flows. Aerosolized PrSBeads in carrier gases of varying oxygen concentrations demonstrated point measurement precisions on the order of 0.1%-1%. A charge-coupled device featuring a double image frame (DIF) feature was used to make spatially resolved pressure measurements within gas phase flows. Errors on the order of 0.5 atm for one standard deviation were demonstrated when 2 x 2 pixel binning (162 x 128 pixel overall resolution) was used, but improved to 0.003-0.005 atm with the use of 32 x 32 pixel binning (10 x 8 pixel overall resolution). Experiments demonstrate the ability to resolve the oxygen concentration differences between a N(2) jet and the surrounding ambient air environment and the ability to measure instantaneous air pressure changes within a square syringe as the plunger is moved in and out. In addition, instantaneous velocity measurements of the airborne PrSBeads in a square syringe were achieved using digital particle image velocimetry at frame rates of 6.4 Hz, thus validating PrSBeads as a tool to simultaneously measure the velocity and pressure within an aerodynamic flow.

Development and characterization of fast responding pressure sensitive microspheres.

The response times of pressure sensitive paint (PSP) and pressure sensitive microspheres to passing shockwaves were measured to investigate their ability to accurately determine pressure changes in unsteady flows. The PSPs tested used platinum tetra(pentafluorophenyl)porphine (PtTFPP), platinum octaethylporphine (PtOEP), and a novel set of osmium-based organometallic complexes as pressure sensitive luminophors incorporated into polymer matrices of dimethylsiloxane bisphenol A-polycarbonate block copolymer or polystyrene. Two types of pressure sensitive microspheres were used, the first being PtOEP-doped polystyrene microspheres (PSBeads) and the second being porous silicon dioxide microspheres containing the novel, pressure sensitive osmium complexes. Response times for the platinum-based PSPs ranged from 47.2 to 53.0 micros, while the osmium-based PSPs ranged between 37.6 and 58.9 micros. For the microspheres, 2.5 microm diameter PSBeads showed a response time of 3.15 ms, while the osmium-based silicon dioxide microspheres showed a response time ranging between 13.6 and 18.9 micros.

Singlet molecular oxygen by direct excitation.

Direct excitation at 1064 nm and detection of singlet molecular oxygen at 1270 nm is made possible by the availability of powerful YAG-lasers and sensitive NIR photomultipliers. Singlet oxygen was generated in condensed phase at 77 K by direct excitation at 1064 nm (without the use of sensitizers). Several luminescing species were observed by time resolved luminescence spectroscopy and luminescence lifetime measurements, including the single molecule (1)Delta(g)and (1)Sigma(g)(+)states as well as luminescence from the [(1)Delta(g)](2) simultaneous transition. As an application we propose a novel method for obtaining quantitative non-intrusive mapping of the 2-D oxygen concentrations and pressure at cryogenic temperatures, which is of importance in aircraft design for high altitudes.

Measurement of respiration rates of Methylobacterium extorquens AM1 cultures by use of a phosphorescence-based sensor.

Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.

Continuous measurement of oxygen consumption by pancreatic islets.

The rate of oxygen consumption is an important measure of mitochondrial function in all aerobic cells. In pancreatic beta cells, it is linked to the transduction mechanism that mediates glucose-stimulated insulin secretion. However, measurement of oxygen consumption over long periods of time is technically difficult owing to the error resulting from baseline drift and the challenge of measuring small changes in oxygen tension. We have adapted an ultrastable oxygen sensor based on the detection of the decay of the phosphorescent emission from an oxygen-sensitive dye to a previously developed islet flow culture system. The drift of the sensor is approximately 0.3%/24 h, allowing for the continuous measurement of oxygen consumption by 300 islets (or about 6 x 10(5) cells) for hours or days. Rat islets placed in the perifusion chamber for 24 h were well maintained as reflected by membrane integrity, insulin secretion, and oxygen consumption. Both acute changes in oxygen consumption as induced by glucose and chronic changes as induced by sequential pulses of azide were resolved. The features of the flow culture system--aseptic conditions, fine temporal control of the composition of the media, and the collection of outflow fractions for measurement of insulin, and other products--facilitate a systematic approach to assessing metabolic and functional viability in responses to a variety of stimuli. Applications to the measurement of effects of hypoxia on insulin secretion, membrane integrity, and the redox state of cytochromes are demonstrated. The system has particular application to the field of human islet transplantation, where assessment and the study of islet viability have been hampered by a lack of experimental methods.

Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry.

A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b595 and b560. The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx. 5 microM). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.

The Calorimetric Detection of Excited States.

Calorimetric techniques offer the photophysicist and photochemist the opportunity to measure a number of parameters of excited states which may be difficult to obtain by other techniques. The calorimetric strategy seeks to measure the heating of a sample resulting from radiationless decays or chemical reactions of excited states. Heating is best measured through volume and pressure transducers, and four calorimeters based on these are described. With calorimetric instrumentation one can perform measurements on samples in the gas, liquid and solid phases over a wide temperature range. Moreover time dependent processes with time constants ranging from microseconds to seconds are amenable to study. Examples of the application of calorimetric techniques to the determination of quantum yields of fluorescence, triplet formation and photochemistry are given.