We describe a homogeneous competitive model immunoassay for determination of thyroxine by multi-frequency phase-modulation fluorescence. Using a non-radiative energy transfer transduction mechanism, B-phycoerythrin conjugated to thyroxine is the energy donor and a carboxymethylindocyanine dye conjugated to anti-thyroxine antibody is the energy acceptor. Energy transfer from B-phycoerythrin to the acceptor results in a decreased lifetime and/or phase angle. The fluorescence lifetime change reflects the extent of energy transfer. In the competitive immunoassay format, the donor-thyroxine conjugate and an analytical sample of thyroxine compete for acceptor-antibody binding sites, resulting in a phase angle change which is dependent on the amount of thyroxine in the sample. Dose response curves of phase angle versus thyroxine concentration demonstrate a broader dynamic range than comparable steady state intensity curves. Since phase-modulation lifetime measurements are largely independent of total signal intensity, sources of optical interference are minimized. The potential for whole blood measurements exists since the energy transfer lifetime method can be extended to longer wavelengths.

Using an evanescent wave fiber optic-based biosensor developed at Naval Research Laboratory, ricin toxin can be detected in the low ng/ml range. Sensitivity was established at 1 - 5 ng/ml using a two-step assay. The two-step assay showed enhanced signal levels in comparison to a one-step assay. A two-step assay utilizes a 10 minute incubation of an immobilized affinity purified anti-ricin antibody fiber optic probe in the ricin sample before placement in a solution of fluorophore-labeled goat anti-ricin antibodies. The specific fluorescent signal is obtained by the binding of the fluorophore-labeled antibodies to ricin which is bound by the immobilized antibodies on the fiber optic probe. The toxin can be detected directly from urine and river water using this fiber optic assay.

Quartz fibers coated with acetylcholinesterase (AChE) or antibody (Ab) are used as biosensors utilizing total reflectance fluorescence for the rapid detection of pesticides. The enzyme biosensor was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ AChE on quartz fibers. The fluorescent signal was generated by hydrolysis of acetylcholine (ACh) that is present in the perfusate. Organophosphate (OP) and carbamate anticholinesterase (AntiChE) insecticides inhibited AChE and reduced the fluorescent quenching resulting from AChE hydrolysis. A parathion biosensor was constructed by immobilizing casein-parathion on the quartz fibers, that bound rabbit antiparathion antibody. The optical signal was generated by perfusing the fibers with fluorescein-labeled goat antirabbit IgG. Free parathion inhibited the binding of antiparathion Abs and reduced the optical signal and provided the basis for detection of parathion. Another immunosensor developed detected the herbicide Pursuit^R by utilizing the reversible binding of a fluorescein-Pursuit derivative to antiPursuit Abs immobilized on the fiber. Unlabeled Pursuit competed effectively and displaced the bound fluorescent compound in a dose-dependent manner. The sensor discriminated effectively between Pursuit-like and structurally unrelated herbicides. The immunosensor offers the advantage of continuous monitoring, ease of operation, speed of detection, low cost, stability, specificity, matrix transparency, and reusability.

Phosphorescence anisotropy measurements have been proposed as a method of following immunological reactions. To assess the utility of the technique, bovine serum albumin (BSA) has been employed as an antigen and its motion detected by covalently labelling the protein with the triplet probe, eosin-5'-isothiocyanate. A monoclonal IgG anti-BSA antibody was added to a solution of the labelled antigen thereby creating a heterogeneous population of rotating species of different sizes. The rotational mobilities of the labelled antigen and the labelled antigen-antibody complex have been examined by measurement of time-resolved phosphorescence depolarization of the probe, excited by a brief laser pulse from a frequency-doubled Nd:YAG laser. The orthogonally-polarized emission components were collected simultaneously and the time-resolved phosphorescence anisotropy (r(t)) calculated using a Marquardt curve fitting procedure. In the initial experiment, the time-resolved anisotropy from each antigen-antibody mixture was found to consist of more than one exponential decay process. The initial anisotropy values r(O) were found to be population dependent. The initial anisotropy values r(O) obtained from the phosphorescence decay measurements were relatively small (less than 0.07), however it can be enhanced by conjugating one of the species to a relatively large polymer bead. The experimental results indicate that the time-resolved phosphorescence depolarization could be exploited to study the kinetics of antigen and antibody interaction. There is also scope for development of the method for optical immunoassay.

A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10^-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

We have explored the use of Merocyanine 540 as a reporter fluorophore in an optical fiber sensor for general anesthetics. Depression of the freezing point of phospholipid vesicles caused by the addition of small hydrophobic molecules, such as general anesthetics, is monitored using an order-sensitive fluorophore. This reagent is immobilized at the tips of optical fibers for remote spectroscopy. Previously, the fluorophore Laurdan was shown to be a sensitive probe of lipid fluidity. However, Laurdan absorbs and emits in the ultraviolet region of the spectrum; problems associated with UV sensor operation include intrinsic/natural absorption and fluorescence, increased risk of tissue damage, low transmission in glass and plastic optical elements, and unavailability of convenient optical fiber accessories. Merocyanine 540 absorbs most strongly around 540 nm and thus eliminates many of the practical problems associated with UV operation. Preliminary studies suggest that this dye is sensitive to lipid bilayer fluidity, although the mechanisms for this sensitivity appear to be based on concentration-dependent aggregation and on the inner filter effect. Because it is highly susceptible to photobleaching, merocyanine 540 is unsuitable for this application.

Fiber optic sensors for various chemical species are well known in the literature, the majority of which are based on changes in fluorescence intensity or intensity ratios due to the presence of the analyte. Recently, several workers have demonstrated analyses based on fluorescence lifetimes. Fluorescence lifetime measurements were performed through a single optical fiber using a modified phase fluorometer. Results are shown indicating that pH could be accurately measured under these conditions with commercially available indicators and instrumentation. The basis of lifetime sensing in the frequency domain is discussed, together with the origin of the very large dynamic ranges achievable with the technique: greater than 100,000-fold analyte concentration range using a single indicator without multiple equilibria.

The method of preparation of a novel plastic thin film sensor which incorporates the fluorescent dye, 8-hydroxy, 1,3,6 pyrene trisulfonic acid is described; the shelf-life of the film is over 6 months. The results of a study of the equilibrium response of the sensor towards different levels of gaseous CO₂ fit a model in which there is a 1:1 equilibrium reaction between the deprotonated form of the dye (present in the film as an ion pair) and the level of CO₂ the film is exposed to. The 0 - 90% response time of the film when exposed to an alternating atmosphere of air and 5% CO₂ is typically less than 3 s. The response of the film towards a random variation in %CO₂ as a function of time compares favorably with that observed at the same time using an infra-red detector.

Most of the disadvantages that exist with electrochemical devices (e.g., short lifetimes, difficult to miniaturize, need of reference electrodes) can be avoided by using optical sensors. Smock et al. describe a device incorporating a ninhydrin coated fused silica rod that could detect ammonia vapor at concentrations below 100 ppb, however, the reaction is irreversible. Guiliani et al. describe a reversible sensor using a dye coated capillary tube. The dye utilized is oxazine perchlorate, a laser dye. They report that the presence of water vapor is an important factor in the detection of ammonia, and the concentration of water vapor must be controlled. Optical sensors built-up in integrated-optic technique with planar waveguide configurations allow the construction of optical sensor systems for a parallel detection of several chemical species, provide the generation of reference signals, and facilitate the problem of cross-sensitivities. Here, we report on integrated-optic sensors for ammonia detection with a sensitivity in the ppb-range. The reaction is reversible, and the response is independent of the water vapor concentration in the test gas.

An adsorption-luminescent sensor enables us to discriminate and determine the vapor of odor substances by measuring temperature dependence of luminescence from the sensor (powder of (gamma) -Al₂O₃) which previously adsorbed odor vapor in the air. Profiles of the adsorption luminescence (AL) spectra as a function of temperature for butanol, acetone, and n- butyric acid are apparently different from each other, and these vapors of 1 to 100 ppm in concentration in the air can be determined from the total luminescence intensity. The study of photoluminescence (PL) observed for the sensor after contact with these odor vapors implies that AL is peculiar chemiluminescence during catalytic oxidation of odor vapor which produces water and surface hydroxide.

The techniques for specifying the angular distribution of luminescent and paramagnetic probes on biological assemblies have been combined in the investigation of probe orientation and order of labeled myosin cross-bridges muscle fibers. This combination has been accomplished on two levels involving: (1) a mathematical formalism that permits the combination of data from individual luminescent and paramagnetic probes, and (2) the introduction of a family of specific extrinsic probes that are capable of producing an interpretable luminescent and paramagnetic signal when attached to a muscle fiber. The mathematical formalism has been applied to several probes of the myosin cross-bridge in muscle fibers to establish that the cross-bridge rotates during muscle contraction to produce muscle shortening (Burghardt & Ajtai, 1992 Biochemistry 31, 200; Ajtai et al., 1992 Biochemistry 31, 207). The luminescent/paramagnetic probes have also been employed in the investigation of order and orientation of cross-bridge in muscle fibers (Ajtai & Burghardt, 1992 Biochemistry 31, 4265). The properties of these dual nature probes invites further development of experimental techniques exploiting the high orientation sensitivity of paramagnetic probes with the ability of the probe to absorb and emit light. Flash-photolysis electron paramagnetic resonance is one such technique that may prove useful in the investigation of probe order in biological assemblies.

Until quite recently the ability to detect and discriminate aerosolized micro-organisms at long range using the laser induced fluorescence (LIF) technique has met with limited success. The lasers which met our logistic requirements had insufficient energies to propagate through the troposphere and excite a target organism. The detectors, though sensitive enough, did not allow us to see a spectral distribution of the fluorescence return. Advances in laser and detector technology has now brought us higher energy, solid state lasers, and very sensitive array detectors. Using this new technology we built and tested an ultraviolet LIDAR against various interferents and a micro-organic contaminant. In this paper we describe the system and method used to detect and discriminate an aerosolized micro-organism at ranges up to 3 kilometers, and the results of this effort.

The dependence of total fluorescence escape from a homogeneous, semi-infinite tissue on tissue optics has been examined. A measurement of total fluorescence escape (F) is related to the intrinsic fluorescence coefficient ((Beta) ) by a factor that depends on the optical properties of the tissue: F((lambda) ) equals (Beta) ((lambda) _x, (lambda) ) (integral) _O^(infinity ) (Phi) (z; (lambda) _x) E(z; (lambda) ) dz. (Beta) is the product of fluorophore absorption coefficient and fluorophore quantum yield. (Phi) is the fluence rate of excitation light ((lambda) _x) in the tissue. E is a dimensionless function that describes the probability for escape to the surface of emission light ((lambda) ) originating at a depth z. The source strength of fluorescence emission at depth z is the product (Beta) (Phi) . Accurate expressions have been developed for the depth dependence of (Phi) and E, valid for both absorption-dominant and scattering-dominant light wavelengths in a tissue. These expressions have been based on Monte Carlo simulations of photon transport. They have been used to evaluate the integral in the above equation to yield an analytic expression for fluorescence escape which is a function of the total diffuse reflectance (R_d) and optical penetration depth ((delta) ) at both the excitation and emission wavelengths. Tissue phantoms have been used to experimentally verify the theory of total fluorescence escape. Non-invasive measurements of total fluorescence escape, diffuse reflectance, and penetration depth allow determination of the intrinsic fluorescence spectrum.

Considerable attention has been given recently to the design and development of nonradiative methods of recognizing DNA in a sequence-specific manner. Earlier, we reported that Ru(bpy)₂dppz²⁺(bpy equals 2,2'-bipyridine, dppz equals dipyrido[3,2:a-2',3':c]phenazine) shows no luminescence in aqueous solution, but upon intercalation into double-helical DNA, bright photoluminescence is observed (A. E. Friedman, et al., J. Am. Chem. Soc., 1990, 112, 4960). Based upon this observation, a sequence-specific molecular light switch has been designed in which a dppz complex of ruthenium(II) is tethered onto an oligonucleotide. An oligonucleotide modified at its 5 foot end has been constructed by coupling the sequence 5'-H₂N(CH₂)₆AGTGCCAAGCTTGCA-3' to Ru(phen')₂dppz²⁺ (phen' equals 5-amido-glutaric acid-1,10-phenanthroline). Like the parent complex Ru(bpy)₂dppz²⁺, the single-stranded metal-oligonucleotide conjugate shows little detectable luminescence in aqueous solution. Addition of the complementary strand results in intense photoluminescence; time-resolved studies show that the emission is biphasic with excited state lifetimes of 500 (60%) and 110 (40%) ns. As expected, addition of a non- complementary strand produces no luminescence enhancement over that of the single-stranded metal-oligonucleotide. These results demonstrate that this oligonucleotide derivatized metal complex can be used to recognize and target specific sequences on DNA, a valuable feature which may lead to interesting and novel applications in hybridization technology.

Biological specimens are characterized by high light-scattering and background fluorescence which cause decrease in sensitivity of immunoassays. These unfavorable effects can be minimized by selecting a label with high absolute sensitivity, appropriate excitation and emission wavelengths, and time-resolved mode of measurements. This can be achieved by the use of metalloporphyrin labels, microsecond logic for discrimination of scattered light and background fluorescence which arises in optical elements, cells, solvents, and samples. The intensity of metalloporphyrin phosphorescence depends to a great extent on pH. Nonionic and cationic micelles have a strong effect on the phosphorescence quantum yield of Pd- coproporphyrin. Under optimum conditions Pd-coproporphyrin can be detected at a concentration of 10^-13M by using modified Arcus 1230 (Wallac, Finland). On the basis of these results a novel class of luminescent labels --phosphorescent metalloporphyrins -- was applied to immunoassays. Pd- and Pt-coproporphyrins were used for the covalent labeling of antibodies and antigens. Special derivatives of a porphyrin with activated side chain were synthesized. Techniques for covalent coupling of porphyrins and their metal derivatives with proteins were developed as well as methods for purification of conjugates. A solid-phase time-resolved porphyrin phosphorescence immunoassay test was developed. The well known `sandwich' and competition techniques are compatible with the proposed method. Two or more metalloporphyrins with distinguishable phosphorescent parameters may be used for the simultaneous determining of several antigens in one sample.

A new time-resolved fluorescence measurement system has been developed. A compact streakscope is used as a detector, and the combination of a spectrograph and a streakscope enables the instrument to measure the fluorescence lifetime and spectrum simultaneously using the single photon counting method with a time resolution of 15 ps (5 ps with fitting software). The dynamic range is achieved up to 105. The instrument response function (IRF) of a streakscope is nearly Gaussian, and it has no tail and no `after pulses' which are observed at the IRF of a photomultiplier used in the time correlated single photon counting system (TCPC). So, a short lifetime of several ten picoseconds can be measured with accuracy. In addition, two dimensional (time and wavelength) single photon counting can be performed without scanning the wavelength, so time resolved spectrum can be measured in a short time with a high S/N ratio.

In this paper we describe the state of development of a flow-through immunosensor for quasi- continuous monitoring of pesticides, especially triazine herbicides, in drinking water. Starting with time-resolved fluoroimmunoassays using Eu-chelates as fluorescent labels, we obtained a detection limit of 0.1 (mu) g/l for some triazine herbicides. The experimental parameters applied in assays on microtiter strips were transferred and adjusted to small carrier beads as solid phase in affinity columns. For fluorescence detection in the FIIA a pulsed laser was used as an excitation source. The light was coupled into a flow-through cuvette and the fluorescence signal was recorded with a photomultiplier tube working in a gated detection mode.

The application of Langmuir-Blodgett films of antibodies as sensitive elements of biosensors based on the TIRF and plasmon resonance principles requires data on the regularity of the monolayers and activity of the antibodies forming these monolayers. The monolayers of the monoclonal anti-Pd coproporphyrin antibodies, carrying a europium label, were formed on water/air boundary by the Langmuir-Blodgett method. Films were then deposited onto the surface of a plate, modified with a thin layer of different siloxane polymer carriers for covalent attachment of the protein monolayer. We used a methodological approach for protein films investigation that consists in monolayer parameters evaluation by means of registration of europium-labeled antibodies fluorescence that form the film. Under surface pressure values the 30 - 35 mN/m (the area for IgG molecule in the film being 6000 - 4000 A²) film takes on tight packing peculiar for Langmuir-Blodgett films. The distribution coefficient between the water-air interface and the subphase for the antibodies at different surface pressures, temperatures, and pH values was measured. The range for the distribution coefficient is 0.02 - 0.01 for IgG molecules. The immunological activity of IgG molecules and steric hindrances for antigen binding in the LB films were estimated by measurement of the intensity of the phosphorescence of Pd-coproporphyrin conjugated with proteins of various molecular weight. The LB films of monoclonal antibodies to Pd-coproporphyrin retained 90% immunological activity for Pd-coproporphyrin (MW 760) binding, 17% binding of Pd- coproporphyrin conjugated with SIT (soybean inhibitor of trypsin, MW 25,000), and 0.44% binding of Pd-coproporphyrin conjugated with TG (thyroglobulin, MW 669,000).

This article describes the development and clinical evaluation of two-site immunometric assays for ferritin, thyrotropin (TSH), and tumor necrosis factor alpha (TNF(alpha) ) using time resolved fluorescent measurement with streptavidin-europium (STAV-Eu⁺³) as a label. The liquid phase antibodies were labeled with amidocaproylbiotin-N- hydroxysuccinimide ester. All three assays were based on microtiterplate technology and could be completed within a working day (incubation times less than 4 h). The ferritin assay was compared with luminescent and enzyme labeled assays using identical components. The TSH assay was compared with a commercial immunoluminometric assay whereas the TNF assay was unable to be compared with another method, only with standards from an independent source. The performance data was excellent with lower detection limits for TSH from < 0.003 mU/l and for TNF(alpha) under 10 ng/l. Intra-assay precision was acceptable within the range of interest with TSH < 4% (0.2 - 50 mU/l), TNF(alpha) < 15% (70 - 8000 ng/l and ferritin < 8% (10 - 500 g/l). Inter-assay precision was < 6% for TSH, < 16% for TNF(alpha) and < 8.5% for ferritin. All assays were performed using commercially available components and proved suitable for routine use.

Molecular mechanics calculations have been applied to the structure determination of 8- coordinate tris(1,3-diketonate) europium complexes and new Eu-chelating agents. We used the modified version of MM2 program to enable it to deal with atoms coordinated to as many as eight other atoms, and we have added europium parameters to the force field. Initial geometries for optimization were obtained from the reported crystal coordinates. In view of the dominance of steric effects on the geometry of the europium complexes we chose to eliminate the bending terms for all bond angles involving the europium as the central atom. Instead we allowed the specific inclusion of 1,3-nonbonded interaction between those atoms bound directly to the metal atom. Geometry optimization for three known europium complexes afforded structures that are in reasonable agreement with x-ray crystal structures.

About 1-2% of the basal human O₂-consumption is taken up by the skin surface. This amount varies depending on the local skin blood flow. A new sensing device, the O₂-flux- optode, has been developed which should allow the measurement of local O₂-uptake of the skin and thereby indirectly its local circulation. This luminescence-based-sensor (1) is permeable for the analyte O₂, (2) is flexible for covering larger areas of the surface, and (3) does not consume the analyte O₂. The new O₂-flux-optode consists of an indicator layer with an embedded oxygen sensitive fluorescence dye and a test layer with known diffusion properties. If this sensor is applied to the skin, the pO₂ between skin surface and test layer is measured. With defined (or measured) pO₂ of the environment (e.g., air) the O₂-flux (J_O(2)) into the tissue can be determined from the pO₂ gradient ((Delta) pO₂) across the test layer and its material properties (C). This yields to the equation: J_O(2) equals C ^* (Delta) pO₂.

A newly developed category of signal processing scheme for the detection of fluorescence lifetime is discussed, with application to a number of inexpensive, compact, and reliable fluorescence lifetime-based fiber optic sensors. With phase-sensitive detectors being used to achieve a high noise-suppression ability, the schemes discussed here include some of the features of existing phase and modulation techniques for lifetime measurement. However, they differ from them in several aspects, especially in that they operate in a way rather similar to that of a phase-locked loop. Based on such schemes, the measured lifetime is converted to a repetitive signal whose period is directly proportional to it, and thus a high measurement resolution can be achieved. Such schemes can be operated over a wide consecutive measurement range of fluorescence lifetime, although they have been developed for comparably low bandwidth (sub-MHz) use for temperature sensor applications using long lifetime (> 1 microsecond(s) ) fluorescent media. Illustrations of the applications of these schemes in lifetime-based fiber optic thermometers are presented showing their promising potential for both biomedical and industrial uses.

Monomolecular thin solid films containing water-soluble Pt- and Pd-porphyrins were prepared and transferred onto solid supports. Surface active charged polymer on the basis of alkylated polyethyleneimine was used to improve extraction of the dye from aqueous solution to the interface. The solid-state materials obtained which showed intense long-decay luminescence were studied by optical methods with emphasis to luminescence quenching and lifetime measurements. The results were applied to quenched-luminescence lifetime-based sensing of relative air humidity. A corresponding fiber-optic prototype device was developed.

Pd-coproporphyrin I (Pd-CP) has optimum phosphorescence characteristics for application in immunoassay. The aim of this study is to work out a universal phosphorescence immunoassay method (UniPhIA) using monoclonal antibodies to Pd-CP and conjugates of various proteins with Pd-CP for detection of insulin. Pd-CP and monoclonal antibodies obtained allow a convenient method for determination of various antigens to be developed, which combines the universal character of the amplified enzyme-linked immunosorbent assay (a-ELISA) with a high sensitivity and the simplicity of the time-resolved phosphorescence immunoassay.

A thermoluminescent sheet (TL sheet) for in vivo measurement of spatial dose distribution near small radioactive sources (mm) and its digital readout system have been developed. The TL sheet, the size of which is 40 cm X 50 cm X 0.2 mm, is a teflon homogeneously mixed with BaSO₄:Eu doped powder of about 5 micrometers . This thin TL sheet is very flexible and can be cut to the desired size and form. The sheet was insensitive to room-light, so it is easy to handle. The sheet was found to have a linear response with a very wide dynamic range from at least 0.002 cGy to 5000 cGy absorbed dose. The spatial resolution of the TL sheet was 62 micrometers corresponding to 8.1 line pairs/mm. These are suitable to measure the in vivo spatial dose distribution near sources because large dose gradients exist over a small area. For intracavitary radiation therapy of a rectal cancer, the in vivo dose distribution on the surface of the cancer was determined with the TL sheet. This TL sheet may provide a convenient means of measuring the dose distribution of various patterns in radiation therapies.

A phase-sensitive flow cytometer has been developed to resolve signals from heterogeneous fluorescence emission spectra and quantify fluorescence decay times on cells labeled with fluorescent dyes. This instrument combines flow cytometry (FCM) and fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on single cells in flow, while preserving conventional FCM measurement capabilities. Stained cells are analyzed as they pass through an intensity-modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using a collecting lens, a longpass barrier filter to block scattered laser excitation light, and a photomultiplier tube detector. Results have demonstrated: (1) signal phase shift, amplitude demodulation, and average measurement of fluorescence lifetimes on stained cells; (2) a detection limit threshold of 300 to 500 fluorescein isothiocyanate (FITC) molecules equivalence for excitation frequencies 1 to 30 MHz; (3) fluorescence measurement precision of 1.3% on alignment fluorospheres and 3.4% on propidium iodide (PI)-stained cells; (4) the resolution of PI and FITC signals from cells stained in combination with PI and FITC, based on differences in their decay lifetimes; and (5) the ability to measure single decay times by the two-phase, phase comparator, method.

A fiber optic thermometer based on the lifetime of Cr:LiSAF fluorescence, induced by visible light, is reported for applications in the monitoring of temperature during clinical heat treatment on Benign Prostatic Hyperplasia. As the heat source for such treatment stems from an externally applied radio frequency (rf) field (434 MHz), unlike the current technology which uses thermocouples as the temperature sensing element, the use of such a fiber optic thermometer, which is immune to rf interference, allows for the continuous monitoring of temperature and hence further enhances patient safety. In addition, the application of an un- pulsed external rf field as the heat source (necessary with the use of thermocouple-based monitoring), also simplifies the operating of the treatment. A newly developed effective technique for the detection of the fluorescence lifetime for temperature measurement is used, together with the application of a red (670 nm) diode laser as the excitation source, to produce a compact and inexpensive device for this lifetime-based instrument. The temperature dependence of Cr:LiSAF fluorescence lifetime is discussed, with an empirical formula, which can be used for the calibration of the thermometer being presented to describe such dependence. The thermometer covers the temperature region 10 - 100 degree(s)C, with response time less than one second and a satisfactory accuracy under laboratory test conditions is achieved.

A real time frequency domain phosphorimeter capable of measuring precise and accurate excited state lifetimes for determining oxygen is described. This frequency domain instrument does not make use of cross correlation techniques traditionally used in frequency domain fluorometers. Instead, the electrical signal from the detector is filtered to contain only the first several harmonics. This filtered signal is then sampled and averaged over a few thousand cycles. The absolute phase and absolute modulation of each sampled harmonic of the excitation and of the luminescence is computed by employing fast Fourier transform algorithms. The phase delay and the modulation ratio is then calculated at each harmonic frequency. A least squares fit is performed in the frequency domain to obtain the lifetimes of discrete exponentials. Oxygen concentrations are computed from these lifetimes. Prototypes based on these techniques were built employing commercially available components. Results from measurements in saline solution and in the arterial blood of dogs show that oxygen concentrations can be determined reproducibly. The system drift is less than 1% in over 100 hours of continuous operation. The performance of fiber optic sensors was evaluated in dogs over a period of 10 hours. The sensors tracked changes in arterial oxygen tension over the course of the experiment without instabilities. The overall response of the system was about 90 seconds. The update time was 3 seconds.

Water-soluble polymers are of considerable potential as novel solubilizing media for spectroscopic and photochemical applications. In this paper, we describe a variety of luminescence approaches to the study of the hydrophobic domains created in such species. Techniques discussed include time-resolved anisotropy measurements (TRAMS) room- temperature stabilized phosphorescence (RTSP) and energy transfer.

The application of near-infrared molecules as potential probes for fiber optic applications is discussed. A bis-carboxylic acid derivative of a carbocyanine dye was utilized to determine solution pH changes, and a 2,3-dicyanonaphthalene derivative was used as a probe for the metal concentration studies. The absorbance and fluorescence wavelength of the NIR dyes used in the optical probe are above the 700 nm to minimize interference. The probe dye was entrapped in a polymer matrix attached at the distal end of two optical fibers. The output of a 780 nm semiconductor laser diode beam was focused on one fiber and used for excitation of the probe dye. The second fiber was used to collect the fluorescence from the probe. The fluorescence intensity was monitored as the probe was placed in vials containing the analyte. The optical probe response was found to be about 20 sec during the metal ion measurements; however, the probe response was shorter for pH measurements. The long term stability of the probe was investigated by a week-long period of observation and resulted in a change of about 2% in intensity signal, indicating suitable long term stability.

Benzophenone was used as the photoreactive moiety in the synthesis of several water soluble, fluorescent photoaffinity labels. The following compounds were synthesized: 5-(2-(p- benzoylbenzamido)ethylamino-1-napthalenesulfonate (BzEDANS); 5-(2-(p- benzoylbenzamido)hexylamino-1-napthalenesulfonate (BzHDANS); and 4-benzoyl-1- benzamidofluorescein (BzAF). BzEDANS and BzHDANS were found to be unsuitable for use as photochemical probes. They were incapable of photoinduced covalent binding to methylene carbon due to intramolecular triplet-triplet energy transfer. BzAF was synthesized because its fluorescent moiety, fluorescein, is an inefficient acceptor for intramolecular quenching of the benzophenone triplet state diradical intermediate. BzAF was found to be a suitable and efficient photolabel and is presently a prototype for a new class of fluorescent photolabel.

Macrocylic complexes of the lanthanide (III) ions were functionalized to permit their attachment to antibodies, nucleic acid probes, and any other species capable of specific binding. The Eu(III) complex was found to possess a combination of properties (water solubility, inertness to metal release, ligand-sensitized luminescence, reactive peripheral functionalities) that make it suitable as a luminescent marker for bio-substrates. Its coupling to avidin was achieved, and the properties of the resulting conjugate were investigated.

We have developed a compact and sensitive detection system based on the fluorescence excitation by commercial laser diodes in the VIS and NIR. The system comprises separate excitation, sample, and detection chambers and shows the potential of quantitatively determining immunological and other parameters with small and rugged instruments. Our examinations specifically addressed technical aspects of laser diodes which are responsible for the quality of fluorescence excitation, such as the characterization of the mode structure, the temperature and current dependency of the optical power emission, and the selection and adjustment of the laser wavelengths for optimal overlap with the fluorophors' absorption maxima (namely rhodamine 800, indocyanine green IR-125, and latex-bound dyes). Signal-to- noise level ratios of about 1:1 are reached at sub-nanomolar concentrations in an illuminated sample of 20 (mu) l using a standard photomultiplier tube in analog mode as the detector. Different arrangements of optical filters (e.g., interference, colored-glass type, and polarizing filters) have been optimized with regard to the excitation and detection paths. Furthermore, measures toward an internal calibration of the system have been investigated.

The luminescence emission of singlet molecular oxygen (¹O₂) generated by bacteriopheophytin a, a near infrared emitting photosensitizer, was measured using a new high sensitivity spectrometer system for time and spectral resolved near infrared detection. The instrument uses a low energy pulsed nitrogen laser (40 (mu) J per pulse) to optically excite the photosensitizer and is capable of a time resolution of 40 ns per data point and an instrument response function of 350 ns FWHM (full width at half maximum). The use of a low energy (and relatively low cost) source provides sufficient system sensitivity to measure time resolved spectra in the near infrared with high spectral and temporal resolution. The simultaneous detection, with high accuracy and repeatability, of both the temporal and spectral dependence of the photo-processes of ¹O₂ generation, especially with near infrared emitting photosensitizers, may further stimulate the current intensive investigations concerning the activity of ¹O₂ to bio-molecules.

A method is described that makes it possible to measure luminescence decays for both parallel and perpendicular excitation geometries in a single experimental run. The rotational diffusion of oxazine 725 in five solvents of different viscosities is characterized using the new procedure. The measurements are challenging in that the rotational diffusion times are close to the excited state lifetime. The advantage of the new procedure lies in the increased confidence that comes with measuring both polarizations under the same conditions. The same principle is applicable in any circumstance involving the comparison of two or more samples.

An infrared fluorescence microscope consisting of a laser diode for exciting infrared fluorophores attached to DNA oligo-nucleotides and a silicon avalanche photodiode for detecting the infrared emission has been designed. The microscope was mounted on a scanning platform which could be optimally focused on an electrophoretic gel (0.1 - 0.4 mm thick) sandwiched between two glass plates. Background fluorescence is minimal in the infrared region of the optical spectrum. In addition, the optics were designed to further minimize this background fluorescence while maximizing the signal output. A 5 pM fluorophore-DNA concentration in unpolymerized gel solution (about 2000 molecules in an irradiated volume of 600 pL) gave a signal-to-noise ratio of 4:1, 3:1, and 2:1 for a glass-gel-glass sandwich made using quartz, borosilicate, and soda-lime glass, respectively.

In order to simplify a laser fluorometric detector for capillary electrophoresis a semiconductor laser emitting at 660 nm is used as an exciting source. A labeling reagent fluorescent in the deep-red region is newly synthesized and used for determination of amino acids. The separation resolution is several hundred thousand and the detection limit is subattomole levels. Protein is also labeled by the dye developed and is measured at subfemtomole levels. Contrarily, nonfluorescent samples are detected by indirect fluorometry. Amino acids, flavin adenine dinucleotide (FAD), and deoxyadenosine monophosphate (dAMP) are determined, the detection limit being subpicomole levels.

Spectrofluorometers employing xenon arc lamp excitation and photomultiplier tube detectors afford sensitivity over the UV/VIS spectral region for which these instruments were designed, but suffer sensitivity limitations in the short-wave near infrared (NIR) region (800 - 1000 nm) because of their limited source energy and low detector quantum efficiency. To achieve high sensitivity in the NIR region, a 30 mW diode laser source, an imaging spectrograph, and a cryogenically cooled charge-coupled device (CCD) have been combined in a spectrofluorometer specifically designed for use in the NIR region. The diode laser source incorporates integral source filters, optics, and a beam trap, and utilizes a vertical beam geometry which provides an illuminated volume oriented conveniently for the imaging of fluorescence emissions on the entrance slit of the spectrograph. Data is presented which demonstrates that the temporal and spectral stability of the source is equal or superior to that of an arc lamp for solution-phase fluorometry. In addition to spectral information, the CCD detector provides spatial resolution of fluorescence emissions along the vertical path of the excitation beam. An absolute photometric calibration of the CCD detector, and measurement of its read noise, fixed pattern noise, and linear dynamic range is performed using the photon transfer technique of Janesick, et al. Improvement in the instrument performance by more than six decades is demonstrated by measured LOD of NIR dyes using a commercial SLM 4800 instrument and the new diode laser/CCD arrangement. Origin of the present detection limits is discussed.

A number of measurement schemes for the determination of dissolved or gaseous oxygen have been reported, most of them based on fluorescence quenching methods. They have the disadvantage of requiring large and heavy instrumentation and, therefore, are not suitable for micro-integrated technologies. As a result, the applicability is greatly limited. We introduce a system based on semiconductor devices (LEDs, photodiodes, low cost analogue and digital components) which is well suited for hybrid solutions, and represents a realistic alternative to existing micro integrated electrochemical probes. New LED-compatible sensor membranes were developed and characterized. The influence of straylight on the overall transfer function of the sensor system was investigated and possibilities for reduction or even elimination of this influence are presented. The overall performance of the instrument in terms of sensitivity, detection limits, long-term stability, and reproducibility is presented. The system was applied to the measurement of dissolved oxygen in drinking water and sea water.

We have investigated the use of micron-sized liquid droplets as sample medium to detect single fluorescent molecules in solution. The use of microdroplets (5 - 15 micrometers diameter) offers several powerful advantages over single-molecule detection schemes involving measurements on bulk liquids where the probe volume is defined by the laser beam. In addition, cavity-quantum electrodynamical (QED) effects have been observed which influence both spontaneous emission rates and fluorescence yields of dye molecules in these microspheres.

A picosecond single-photon fluorescence lifetime instrument has been constructed for sensor development using liquid light guide coupling. The performance and application of the instrument are demonstrated with respect to examples of a proposed new type of energy transfer sensor for the detection of metal ions in solution. Novel instrumental features include the capability for performing multiplexed measurements using a distributed sensor network.

The goal of our research program is to develop competitive and sandwich fluoroimmunoassays with high sensitivity and fast response time, that do not require external reagents. Our approach to this problem is to employ an optical immunoassay based on total internal reflection fluorescence (TIRF). Specifically, monoclonal antibodies are immobilized on a planar waveguide. Total internal reflection of light in the planar waveguide sets up an evanescent field which extends about 2000 angstroms from the interface. In the competitive immunoassay, a fluorescent label is coupled to a small synthetic antigen which is packaged with the antibody. In the absence of analyte, the fluorescently labeled antigen binds to the antibody and is excited by the evanescent field. Upon the addition of analyte, the fluorescently labeled antigen molecules are displaced by unlabeled antigen molecules and diffuse out of the evanescent field. In the sandwich assay, a primary or `capture' antibody is immobilized on the planar waveguide, and a secondary or `tracer' antibody (which is labeled with a fluorescent dye) is added to the bulk solution. In the absence of analyte, the tracer antibody remains in solution and very little fluorescence is observed. However, upon addition of analyte, a `molecular sandwich' is formed on the waveguide, composed of: (1) the capture antibody; (2) the analyte; and (3) the tracer antibody. Once this sandwich forms, the tracer antibody is within the evanescent field and fluoresces. Fluorescence emission is detected by a charged- coupled device (CCD). Using this approach, we have developed a prototype immunosensor for the detection of human chorionic gonadotropin (hCG). This device meets our design goals and exhibits a sensitivity of 0.1 - 1 pmolar.

Ionophores which are at the same time selective and sensitive for lithium ion in the presence of physiologically relevant concentrations of sodium, potassium, and hydrogen ions have been the subject of much research for application to ion selective electrodes. We report here our recent work to design, synthesize, and test both neutral and anionic ionophores which respond selectively and reversibly to lithium ion with an increase in fluorescence. The intent is to produce lithium sensitive fluorophores for fiber optic sensing. Selectivities of fluorescent acyclic and crown macrocyclic ionophores are described in acetonitrile and aqueous/chloroform solvent systems.

The effects of some fundamental parameters on the performance of optical immunosensors based on fluorescence induced by an evanescent wave (total internal reflection fluorescence, or TIRF) are evaluated. Both competitive and sandwich systems are discussed and compared. The performance of such devices are described using the range within which the error is less than a fixed value. Methods of controlling the effects of various parameters are considered.

Silicon avalanche photodiodes (APD) have been used for photon counting for a number of years. This paper reviews their properties and the associated electronics required for photon counting in the Geiger mode. Significant improvements are reported in overall photon detection efficiencies (approaching 75% at 633 nm), and timing jitter (under 200 ps) achieved at high over-voltages (20 - 30 V). Results obtained using an active-mode fast quench circuit capable of switching over-voltages as high as 20 V (giving photon detection efficiencies in the 50% range), are reported with a dead-time of less than 50 ns. Larger diodes (up to 1 mm diameter), usable in the Geiger mode, which have quantum efficiencies over 80% in the 500 - 800 nm range also are reported.

UPY-F is a software dedicated to solving various queries issued by end-users of spectrofluorimeters when they come across a problem in the course of an experiment. The main goal is to provide a diagnostic for the nonpertinent use of a spectrofluorimeter. Many artifacts may induce the operator into trouble and except for experts, the simple manipulation of the controls of a fluorimeter results in effects not always fully appreciated. The solution retained is an association between a powerful hypermedia tool and an expert system. A straight expert system offers a number of well-known advantages. But it is not well accepted by the user due to the many moves between the spectrofluorimeter and the diagnostic tool. In our hypermedia tool, knowledge can be displayed by the means of visual concepts through which one can browse, and navigate. The user still perceives his problem as a whole, which may not be the case with a straight expert system. We demonstrate typical situations in which an event will trigger a chain reasoning leading to the debugging of the problem. The system is not only meant to help a beginner but can conform itself to guide a well trained experimenter. We think that its functionalities and user-friendly interface are very attractive and open new vistas in the way future users may be trained, whether they work in research labs or industrial settings, as it could namely cut down on the time spent for their training.

With the aim of obtaining efficient near infra-red emitters, a family of ruthenium and polyruthenium complexes made from tetrapyridyl analogs and terpyridine ligands was synthesized. It was expected that the increase in size of the complex would result in the progressive shift of the absorption and emission spectra towards the infra red and above all that the stability and rigidity of these complexes would be associated to a high emission yield. If the expected shifts are obtained, with emission at 820 nm for a bisruthenium complex for instance, quantum yields are very low, usually between 10^-2 and 10^-3. More disturbing, these compounds display an `anormalous emission' at shorter wavelength. The latter does not appear to be due to impurities since it is different for all these molecules which are built from the same precursors treated in similar conditions. Preliminary studies point to the fact that the bite angles of the multidentate ligands are not adapted to the binding to ruthenium and that this mismatch gives rise to a tension which can be released only by having a ligand pop off one binding position. The resulting species is distorted, with a reduced conjugation or electronic communication between the metal centers, and displays the short wavelength emission. Rigidification of the tetrapyridyl ligand does not improve the emission yield, a fact which is in agreement with the above explanation. Absorption and emission spectra for all these compounds are analyzed.