The near infrared (NIR) fluorescence has several advantages as a detection system over visible or uv fluorescence counterpart. The spectral properties of cyanine dyes offer advantages for detection in the longer wavelength NIR region. The present work focuses on the synthesis of NIR dyes and their applications in bioanalytical chemistry. Several cyanine dyes have been synthesized. The spectroscopic characteristics such as molar absorptivity, fluorescence quantum yield, excitation and emission maxima have been determined. The potential utilities of these new dyes as probe and reporter molecules in bioanalytical methods have been explored. Dyes bearing reactive functional groups such as isothiocyanate, carboxylic acid or hydroxyl groups can be directly used for covalent labeling of proteins and nucleic acids. Their importance in the synthesis of dye labeled phosphoramidites and dye labeled dideoxynucleotides and use in DNA sequencing have been evaluated.

The availability of low cost laser diodes and new fluorescent dyes has made portable biosensors a reality. Previously, we have examined the variation in the fluorescent signal generated in an antigen-antibody reaction when the antigen is labeled with dyes exciting at different wavelengths. In this study, we looked at the effect of changing dyes and wavelengths on a sandwich immunoassay for the F1 antigen from Yersinia pestis, the etiologic agent of plaque. The F1 immunoassay has previously been demonstrated to work in serum, plasma, and even whole blood, when performed using a fiber optic biosensor. In this study, we demonstrated that changing to cyanine dyes enhanced the sensitivity of the detection without altering the immunochemistry of the assay.

A diode laser fluorometer was constructed and evaluated using rhodamine 800, a commercially available laser dye. Rhodamine 800 was evaluated as a precolumn derivatization reagent through an intermediate for conjugation to amine containing analytes. Preliminary results show that the derivative is nonfluorescent. Rhodamine 800 was successfully used as a mobile phase additive for HPLC with indirect detection of valproic acid from plasma. Nile blue, was evaluated as a derivatization reagent for carboxyl containing analytes. A limit of detection of 0.96 pmol was observed for the nile blue conjugate of benzoic acid using a conventional fluorescence detector. A cyanine dye, was used as a near infrared labeling reagent for compounds containing primary amine functionality. The spectral properties of the cyanine derivative of 2-adamantanamine were similar to the unreacted label and very favorable for low level detection.

We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. We show that the asymmetric complex Ru(bpy)₂(dcbpy) (PF₆)₂ displays a high anisotropy value near 0.26 when excited in the long wavelength absorption band, and an intensity decay time near 400 ns. For covalent linkage to proteins, we synthesized the N- hydroxy succinimide ester. We measured the intensity and anisotropy decays of [Ru(bpy)₂(dcbpy)] when covalently linked to proteins and in solutions of increasing viscosity. These data demonstrated that the probes can be used to measure rotational motions on the microsecond(s) timescale, which so far has been inaccessible using luminescence methods. We also used this probe in a fluorescence polarization immunoassay of HSA. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format with unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high molecular weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low molecular weight antigens.

A LI-COR Model 4000 DNA Sequencer has been modified by removing the internal scanning infrared fluorescence microscope and combining it with an external, orthogonal scanner. Due to the reduced background fluorescence and light scattering of nylon membranes in the near- infrared (8000 nm) as compared to the visible region of the optical spectrum, sensitivity of labeled DNA fragments is enhanced. Dot blots of dilution series of labeled oligonucleotides reveal a detection limit of 25 attomole (25 X 10^-18 mole). DNA fragments blotted onto nylon membranes using direct transfer electrophoresis in multiplex DNA sequencing can also be detected and subsequently analyzed.

We report the photon-counting and timing performance of various Single-Photon Avalanche Diodes (SPADs) employed to detect single photons in the near-infrared wavelength range. Suitable Silicon structures achieve high quantum efficiency (70% at 800 nm) and can work up to 1.1 micrometers . Ge SPADs and InGaAs devices are sensitive up to 1.4 micrometers and 1.6 micrometers , respectively, wit ha few percent-quantum efficiencies. We compare these results with the performance of state-of-the-art photomultiplier tubes with extended near-infrared sensitivity. We also report the first results obtained with a germanium quad-cell sensor, which may be considered the first step towards the development of SPAD arrays.

Indirect fluorescence detection of electrically neutral compounds separated by micellar electrokinetic chromatography is performed using a semiconductor laser as an exciting light source. Oxazine 750 is used as a visualizing agent of which absorption maximum is near 680 nm. A surfactant, tetradecyltrimethylammonium chloride, is used to form micelles and to prevent adsorption of oxazine 750 with a positive charge on the capillary wall negatively charged. This surfactant coats on the capillary wall so that oxazine 750 is repulsed electrically on the capillary wall. In this technique, some aromatic compounds with relatively polar functional groups, such as aniline and nitrobenzene, could be separated and detected, while nonpolar compounds such as benzene and toluene can not be detected. The range of the detection limit is from 4.2 X 10^-4 to 1.6 X 10^-3 M (S/N equals 3) for the aromatic compounds. The detection mechanism is based on enhancement of the fluorescence intensity in the micellar solution and on exclusion of the fluorophore attached at the hydrophilic moiety of the micelle by a hydrophilic sample.

Our new developed time-correlated single photon counting electronics allows for continuous registration of time-resolved fluorescence signals in millisecond intervals. With this electronics, adapted fluorescence measurement system, we studied single molecule transitions through a focused laser beam. In our paper we present measurements on single rhodamine 6G- molecules and discuss data processing methods, using the full information of the time resolved measurement. The use of this information influences significantly the reliability of the detection of single molecule transits. Further developments and applications of this method are discussed.

There is now definitive evidence from Raman and X-ray studies that DNA in a relaxed condition in aqueous solution is in the standard B type conformation. On the other hand, DNA in crystals, in concentrated salt solutions and bound to proteins can exist in other configurations. The question arises: what is the structure of DNA in the nucleus of a living cell where its long length must be subjected to repeated folds in order to pack it into such a small space? To answer this question we designed and built a laser Raman microscope to take the Raman spectra of DNA in the packaged state in which it occurs in nature. Spectra have been taken of the DNA in salmon sperm, squid sperm, and the nucleus of the salivary gland of drosophila melanogaster. The majority of the deoxynucleotide residues in the DNA in the nucleus belongs to the B-type family of conformations. However, the DNA in the nucleus may have a different furanose ring pucker than it does in solution.

Raman spectroscopy with CCD detectors and 780 - 830 nm laser provides sufficient sensitivity and freedom from fluorescence to acquire high SNR Raman spectra from breast biopsy tissue. Spectra of normal and diseased tissue differ substantially, and Raman spectrometers are now available in configurations amenable to clinical use.

Near infrared (NIR) Raman spectroscopy provides a powerful method for quantitative histochemistry of human tissue and disease diagnosis. The feasibility and potential of this technique for in situ histochemical analysis of human coronary artery has been demonstrated and presented in other reports from our laboratory. In this work, we review recent results obtained with the NIR Raman spectroscopy on a variety of tissue types studied at the MIT Laser Biomedical Research Center. We have collected NIR Raman spectra from colon, bladder, breast, and carotid artery. For colon, bladder and breast, consistent differences between carcinoma and normal tissue spectra were observed. For colon and bladder, the spectral differences appear to be due to an increased content of nucleic acid in carcinomas, while the spectral changes in malignant breast tissue are associated with an increase of protein content. Spectra from carotid artery have similar features as those from aorta and coronary arteries. We also show some preliminary results obtained with a NIR Raman microspectroscopy setup with 20 micron lateral resolution. The biochemical distributions for normal and diseased regions on the same tissue samples are observed. The potential of using this NIR Raman spectroscopy for detection and characterization of carcinoma and atherosclerosis, is discussed.

In vivo fluorescence measurements were followed by in vitro NIR Raman measurements on 36 human cervical biopsies, 18 with suspected cervical dysplasia. Fluorescence spectra collected at 337 and 460 nm excitation were used to develop a diagnostic algorithm to differentiate between normal and dysplastic tissues. Using a fluorescence algorithm optimized on another much larger data set, a sensitivity and specificity of 80% and 67% were observed for differentiating squamous intraepithelial lesions (SILs) from all other tissues for the 36 samples. In general, inflammation and metaplasia samples were misclassified at SILs in the larger sample set. A sensitivity and specificity of 57% and 100% were achieved for differentiating high grade from low grade SILs using a second fluorescence algorithm developed with a larger sample set. Using Raman scattering peaks observed at 626 and 1070 cm^-1, SILs could be separated from other tissues with a sensitivity and specificity of 88% and 100% in an algorithm optimized on the data set consisting of the 36 samples from this study. In addition, inflammation and metaplasia samples are correctly separated from the SILs. The band at 1656 cm^-1 could differentiate both SILs from all other tissues as well as high grade from low grade lesions with a sensitivity of 91% and 88% and specificity of 86% and 100%, again using an algorithm optimized on the set of 36 samples. However, these encouraging results must be considered preliminary because of the small sample size. Performance of the fluorescence algorithm to differentiate SILs from squamous normals (sensitivity equals 84%, specificity equals 82%) was higher than was observed with a smaller data set when tested retrospectively on a larger prediction set of 237 samples.

The latest generation of solid state detectors including Charged-Coupled Devices (CCD) and Charge-Injection Devices will be reviewed. Over the last several years, these detectors have contributed to major advances in Raman spectroscopy. Compact Raman systems using diode laser excitation, fiber-optic probes, and new generations of imaging spectrometers have been developed. New approaches for implementing very high throughput monochromators will be discussed, and their impact on the overall Raman system considered. Detection sensitivities obtained using these new optical geometries and array detector technologies are truly astounding. Even at an analyte concentration of a few ppm, a spectrum with several identifiable peaks can be obtained in a mater of seconds. This high sensitivity coupled with the ability of fiber optics to provide remote as well as in-situ sampling now make Raman a technique appropriate for many medical, environmental, and industrial applications. An overview of combining proper optical systems with currently available and soon to be introduced detectors will be presented. A variety of operating parameters for today's and tomorrow's arrays including readout noise, quantum efficiency, dark current, cosmic ray noise, and readout modes will be considered.

A family of indicators has been developed for fluorescence lifetime-based measurement of oxygen, pH, carbon dioxide, and potassium, all the indicators being derivatives of the same chemical compound and having identical spectral and lifetime properties. The indicators show an absorption accessible to low- cast light sources, a large Stokes shift, and long fluorescence decay time. all indicators can be excited at the same excitation wavelength, monitored at the same emission wavelength, and measured within the same time range. This opens the possibility of building a compact lifetime-based instrument to simultaneously measure blood gases and cations.

Recently, we have demonstrated a fluorescence-based fiber optic biosensor for zinc in aqueous solutions. Binding of zinc to the active site of human apocarbonic anhydrase II is transduced by subsequent binding of a fluorescent inhibitor, dansylamide, to the zinc in situ, resulting in large changes in the wavelength, quantum yield and lifetime of the danslamide emission. These fluorescence changes can be readily measured through optical fiber, and yield subnanometer detection limits and 50 dB dynamic range with excellent selectivity. However, the dansylamide is only excitable in the ultraviolet, a spectral regime where fiber optic attenuation is very high; longer wavelength fluorescent inhibitors akin to dansylamide are not yet available. Thus we chose a different transduction scheme wherein the enzyme is labeled with a suitable fluorescent tag and the inhibitor is colored, absorbing in the visible region. When zinc is bound the inhibitor can then bind, bringing it in close proximity to the fluorescent tag and allowing energy transfer to occur; the energy transfer can be followed by changes in intensity or, preferably, lifetime. Recent results using gas laser and laser diode excitation will be shown.

This paper presents the development of an instrument using solid state components for luminescence lifetime based sensing. For a number of applications luminescence lifetime based sensing is the preferred method because of its inherent referencing possibility. Further, lifetime based instrumentation promises a simplified optical construction, since the measurement is, within certain limits, independent of the signal intensity. Various measurement schemes have been reported, especially for optical oxygen sensors, using dynamic fluorescence quenching as the information carrier. However, most of them require bulky and expensive instrumentation because of the need for high-frequency modulated excitation sources and detection systems. In general photo-multipliers have been required. We report on the development of a measurement scheme using low-cost semiconductor devices (light emitting diodes, photo-diodes). The detection system is based on heterodyne demodulation techniques for reduction of the signal frequency range. The basic principles of the system is described and a comparison with existing measurement schemes is presented. The capabilities of the system are demonstrated with measurements on two sensor types having luminescence lifetimes in the range of 1 microsecond(s) and 50 microsecond(s) . Finally, a custom CMOS integrated circuit is presented which implements the front-end of the detection system.

Fluorescence lifetime-based sensing is now recognized as a valuable methodology in clinical and analytical chemistry. For clinical chemistry, or for non-invasive sensing through skin, it is often necessary to obtain quantitative information in highly scattering media. Lifetime-based sensing can be used to obtain quantitative measurements in turbid media. We describe frequency-domain lifetime measurements in intralipid suspension, and present a reliable technique which allows phase-modulation lifetime measurements of fluorophores dispersed within or localized within intralipid. Lifetimes can be measured using an intensity decay law which accounts for the time delays and pulse-broadening effects of multiple light scattering events occurring in the intralipid. Alternatively, the phase and modulation measurements can be performed relative to a reference fluorophore of known lifetime. This approach provided reliable lifetime data for a pH-sensitive fluorophore contained within a microcuvette 4 mm under the surface of an intralipid suspension.

Fluorescence sensors for transition metal ions are demonstrated using the principle of Forster energy transfer from a fluorophore embedded in a Nafion polymer to a metal ion in aqueous solution. The resonance requirement for energy transfer and hence specificity will be demonstrated for cobalt, copper and uranyl ions. Diffusion of these ions in the polymer matrix was also determined with, a diffusion coefficient for Co²⁺ in Nafion of 4.91 X 10^-7 cm²s^-1 and for Cu²⁺ 7.72 X 10^-7 cm²s^-1.

Multifrequency phase-modulation data for fluorescence lifetime and anisotropy decay standards have been obtained for liquid samples in 96-well microtiter plates. We coupled the bifurcated fiber optic probe of a fluorescence plate reader to a phase and modulation time- resolving fluorometer. Laser excitation was used and multifrequency data could be acquired quickly over a range of modulation frequencies from 15 to greater than 150 MHz, at each well position, through the use of multiharmonic Fourier methods. We evaluated the system's performance for UV-excited fluorescence, with the goal of screening quality or properties of proteins or peptides by steady-state or dynamic intrinsic fluorescence intensity or anisotropy using the high throughput 96-well plate format.

Results are presented on the cross-correlation of the results of an accurate theoretical model with experiment data obtained for YAG:Cr³⁺ used as a temperature sensor, obtained over a wide range of temperature, including the cryogenic and biomedical (approximately 37 degree(s)C) region, and a close correlation is seen. Both sets of results support the hypothesis that the phonon-induced transitions between the ²E and ⁴A₂ states have a significant effect on the lifetime of the YAG:Cr³⁺ fluorescence at low temperatures, where the concept of a single equivalent phonon is proposed to account for the mixing effect of phonons with various energy, and is used to modify the single configurational coordinate model discussed. Typical temperature discrepancies between the theoretical and experimental systems are 0.4 K at 300 K, 0.8 K at 500 K and 0.4 K at 900 K.

Advances in long wavelength fluorophore development have reduced interference from the naturally occurring background fluorescence often present in environmental samples, permitting significant progress in explosive and environmental sensing. The Center for Bio/Molecular Science and Engineering at the Naval Research Laboratory has developed a fluorometric, antibody-based, continuous flow immunosensor which can detect nanogram quantities of small molecular weight molecules such as trinitrotoluene and pentaerythritol tetranitrate. In the flow immunosensor, antibodies are immobilized onto a solid support matrix, exposed to a fluorescently tagged analyte, and placed into a small disposable column connected to an aqueous flow stream. Upon sample introduction, an amount of the fluorescently labelled analyte is displaced that is proportional to the concentration of unlabelled analyte present in the sample. The intrinsic nature of anti-body antigen binding also provides the unit with an inherently high degree of sensitivity and specificity. A positive signal can be generated in under 2 minutes, allowing quick analysis of numerous samples with minimal data handling. Development of a single column assay for the detection of both explosives has decreased both detection time and cost per assay. Cross reactivity studies and studies investigating interference from intrinsic fluorescence in several media have also been conducted.

Four-wave mixing is presented as an ultrasensitive detection method for liquid analytes in capillary flow cells and for circular dichroism measurements. A two-input-beam forward- scattering degenerate four-wave mixing optical setup offers simplified optical alignment, effective use of compact low-power lasers, and convenient interface to powerful chemical separation methods using capillary columns. The use of a single focusing lens for both input beams provides very high wave-mixing efficiency and tight focusing inside a small detection probe volume. Attomole or better detection sensitivity levels of this nonlinear laser detector compare favorably to those of many conventional laser-based analytical methods, yet this nonlinear detector can detect both fluorescing and non-fluorescing analytes. One can conveniently interface this multi-wave mixing detector to liquid chromatography and capillary electrophoresis for on-column detection. Nonlinear wave-mixing as a sensitive `direct' or `indirect' absorbance detection method offers important advantages in many biomedical applications.

A novel assay, Pi Overlapping Ring Systems Contained in a Homogeneous Assay (PORSCHA), has been developed which relies upon the change in fluorescent spectral properties that pyrene and its derivatives show as a function of their proximity. When two pyrene rings are sufficiently close such that their pi-systems can overlap during the excited lifetime, an excimer emission centered at 480 nm is observed. When the pi-systems are too far apart for overlap, only monomer emission is observed at 378 nm and 396 nm. Only Angstrom changes in distances are necessary to switch from excimer to monomer emission. Due to its many degrees of freedom, single-stranded DNA adopts a random-coil conformation in solution. When labeled with two pyrene fluorophores and upon binding to its complimentary strand, the excimer intensity changes because the pyrenes may be either closer together or farther apart, corresponding to the reduced degrees of freedom of the double helix. Because the probe does not disturb the system and no separation steps are necessary before detecting the DNA binding, completely reversible, in situ, detection of DNA is possible.

The non-covalent binding and spectroscopic properties of several near-infrared tricarbocyanine dyes to sonicated calf-thymus DNA is reported. The dyes investigated were diethylthiatricarbocyanine iodide (DTTCI), diethyloxatricarbocyanine iodide (DOTCI) and 1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide (HDITCI), which are cationic and possess absorption maxima at 772 nm, 695 nm and 750 nm, respectively, in DMSO. In buffered aqueous solutions, these dyes demonstrated extensive ground state aggregation as seen by gross changes in the NIR absorption spectra. In the presence of double-stranded DNA (dsDNA), the absorption peak in the NIR was restored, indicative of dye-deaggregation. Inspection of the fluorescence emission spectra revealed enhancement ratios of bound-to-free dye ranging from 4.5 for DOTCI to 128 for DTTCI. Scatchard analyses of the dye-dsDNA complexes showed two linear regions, suggestive of at least two possible binding sites on the DNA. For DTTCI, the binding constants were 2.88 X 10⁴ M^-1 and 4.78 X 10³ M^-1.

A series of fluorescent ion indicators were tested for their spectral response to submicromolar levels of 13 divalent and trivalent metal ions in aqueous solution. Upon binding their target ions, these fluorescent compounds exhibit changes in fluorescence emission intensity that are easily detectable, making them useful for direct the detection of soluble heavy metal ions including Hg²⁺, Cu²⁺, Ni²⁺ and Cd²⁺. The fluorescence response of these indicators to ion binding results from photoinduced electron transfer effects, fluorophore/quencher interactions, fluorescence quenching by heavy metal ions or a combination of these processes. The majority of the indicators we tested bind their target ions reversibly with dissociation constants (K_d) near 1 (mu) M (approximately 1 ppm) and detection limits near 100 nM (approximately 100 ppb) at pH 7. However, several indicators exhibit very high affinity for their target ion; for example, Magnesium Green^TM binds Zn²⁺ with a K_d near 20 nM. All the indicators synthesized and tested are based on water-soluble fluorophores that have high fluorescence quantum yields (from 0.3 to 0.7) and can be excited with an Ar laser, fluorometer or hand- held UV lamp. Furthermore, the excitation and emission spectra of these indicators are insensitive to pH changes over the range of 5 to 10, as well as to high concentrations of K⁺, Na⁺, Ca²⁺ and Mg²⁺. These properties make the indicators useful for the direct measurement of metal ions in solutions, such as biological fluids, sea water and waste streams, that contain high concentrations of salts.

Dye-doped porous silica microspheres can be prepared from liquid solution at temperatures near ambient. Microsphere diameter can be controlled between approximately 5.0 microns to in excess of a millimeter. The resulting microspheres can be attached to the distal end of an optical fiber in which the proximal end is attached to a spectrophotometer. Depending upon the organic species doped into the microsphere, a wide variety of sensing functions are possible. In this paper, the use of microsensors for measuring pH, temperature, and solvent content of aqueous solutions is demonstrated. Potential utility of this type of sensor to heavy metals detection and biomedical diagnostics is also discussed.

The design of these fluorescent PET (Photoinduced Electron Transfer) sensors for protons was based on the Fluorophore-Spacer-Receptor' format which has previously been shown to be effective in the construction of fluorescent sensors for various chemical aspects. This presentation shows the importance of kinetic control factors in the design of fluorescent PET sensors, based on the 4-amino-1,8-naphthalimide fluorophore. Two sets of molecules with opposite connectivities of the `spacer-receptor' assembly to the fluorophore have been studied. (Delta) G<sub>PET</sub> is nearly zero for both sets of molecules. However due to their `push-pull' (pi) -electron system (4-amino donor and 1,8-naphthalimide acceptor) an ICT (Internal Charge Transfer) is set up in the lowest excited singlet state. Due to the creation of this electric field upon photoexcitation acceleration or inhibition of a transiting electron can occur. Molecules with the `spacer-receptor' connected at the 4-amino position display `switching on' of fluorescence upon protonation (factor of 25) as the proton-induced suppression of PET leads to fluorescence as the predominant deactivation pathway. However, molecules with the `spacer-receptor' connected at the 1,8-naphthalimide position show only a very slight reduction of fluorescence upon protonation which is attributed to quenching via an intramolecular hydrogen bonded structure. The presence of hydrogen bond donors near the negative pole of ICT excited states is known to result in fluorescence quenching. These results show the importance of kinetic control factors as well as thermodynamic factors in the design of fluorescent PET sensors.

The spectroscopic properties of transition metal complexes make them good candidates for optical sensors. We have developed a coordination complex of ruthenium (II) that changes its visible spectra as a function of local environment. The coordinating ligands to the ruthenium (II) center are cyanide and dipyridophenazine; the cyanides impart solvatochromic behavior, while the coordinated dipyridophenazine functions as a `luminescent light switch' for nonaqeous local environments. The complex was synthesized according to a photochemical protocol, purified by high pressure liquid chromatography, and characterized spectroscopically and by mass spectrometry.

We suggest a new photophysical principle of response of dye fluorescence spectra to the dynamics of its molecular environment. The branched symmetric dye molecule is excited to the degenerate level, which, being unstable, is relaxing to a low-symmetry isomer with substantial Stokes shift. Thus, solvent-viscosity-dependent ratio of fluorescence intensities of unrelaxed and relaxed forms may be observed. The principle was applied to the dyes with branched three-chain polymethine chromophore. In the ground state they are in propeller-like conformation of symmetry C₃. In the excited-state one of the degenerate lower vacant molecular orbitals becomes occupied, which makes the dye conformation unstable. This results in out-of-plane rotation of the end groups with isomerization in the chain and temporal loss of symmetry. This effect should be observed in fluorescence spectra and result in dual emission from unrelaxed and relaxed states, which we observed in glycerol solutions, but do not see in low-viscous solvents. In biomembranes this effect allows to suggest a simple and convenient method of observation of dynamics in lipid bilayer. In phospholipid liposomes however, only the long-wavelength band is seen, which is probably due to the probe location on the surface, in highly mobile environment.

A short review of our published and unpublished studies and investigations of other authors on the sensitization by dyes of near-IR fluorescence of Yb(III) and Nd(III) in solutions and polymers is given. Yb(III) fluorescence lifetimes in different solvents and rate constants of intramolecular energy transfer from triplet state of Eosin to Yb(III) were measured for such systems. Mechanisms of non-radiative transition in Yb(III) salt solutions is discussed. The sensitization of Yb(III) fluorescence by eosinate methylene blue in PMMA was found to occur. Sensitized near-IR fluorescence of Nd(III) also has been detected under two-photon picosecond excitation in DMSO and under one-photon steady-state excitation in POCl₃ + SnCl₄ through intra- and intermolecular energy transfer respectively.

The fluorescence quenching of molecules by analytes of interest, is a widely employed phenomenon in fluorescence sensing technology. Forster type dipole-dipole energy transfer from dye molecules to transition metal ions, provides a method of monitoring the concentration of these ions with some degree of selectivity. Each metal ion has a different absorption spectrum, hence, in principle it is possible to choose different fluorophores for each metal ion. In the present work, quenching studies of the carbocyanine dye DTDCI by transition metal ions in a viscous solvent and a Nafion polymer matrices are reported. The potential for fabricating near-infrared energy transfer sensors is assessed, particularly with regard to detecting copper ions in solution.

The absorption of high intensity laser light by dye molecular probes may produce non-linear effects. They result in both bleaching and darkening, which depends on the rates of depopulation/repopulation of the ground S₀ and excited S₁ states. It is shown, that the darkening is the characteristic features of fast photophysical and photochemical reactions occurring in the S₁ state. This suggests a very simple method for detection and study of these reactions. The method is tested for photoinduced proton transfer and solute-solvent structural reorganizations in polymethine dyes. The results suggest, that the dye probes exhibiting these reactions may be applied for visualization of protonic gradients, microviscosity and distribution of different substances within the living cells. The method is based on the comparison of light transmission images at low and high intensities. It is extremely simple and fast, a single light pulse may be sufficient to create the image.

The photonic detector is a key device for optical instrumentation and fluorescence detection, along with the associated light source and other electric devices. Research and development for photonic devices is in higher response speed, higher sensitivity, and gate/modulation capability. Here, we introduce the following photonic device recent developments: (1) High sensitive photocathode: recently developed high sensitivity photocathode for low light detection. (2) High speed response: the photonic detectors using micro-channel plate have high speed response (time resolution of 20 psec) suitable for both time correlated photon counting and phase modulation methods. (3) Compact photosensor modules: ultra compact photomultiplier tube with TO-8 type of metal package structure will be introduced. Also, newly developed photon counting module photo-sensor. (4) Gated/modulated image intensifiers and photomultiplier tubes: high speed gated/modulated image intensifier photomultiplier tubes have developed for phase modulation method and/or life time imaging microscopy. We will review the recently developed photonic devices for the items mentioned above in this paper.

We present data of simultaneously time and spectrally resolved measurements with the time correlated single photon counting technique using our new SPC-300 PC plug-in module combined with a 64 multianode PMT from Philips and a specially designed router. The SPC- 300 card records in up to 128 time channels simultaneously with a count rate of up to 5 MHz. In this paper we show the performance of the electronics for single decay curves using a MCP-PMT detector from Hamamatsu (R 3809U) and for wavelength resolved detection using an 8*8 multi-anode PMT from Philips (XP1702). An ultimate instrumental response function (IRF) of 34 ps and a maximum count rate of 300 000 cps was achieved by using the ultrafast MCP-PMT and a subtractive double monochromator. By using the multimode PMT coupled to a polychromator we got an IRF of 800 ps at 2.3*10⁶ cps. The fluorescence signal was recorded at 8 different wavelengths simultaneously. For the test measurements we used pure Fluorescein, Rhodamin 6G and DODCI solution as well as a mixture of Fluorescein and DODCI. We got an excellent distinction between the two species. The decay times (3.9 ns, 1.1 ns) are in good agreement with the single curve measurement at a fixed wavelength.

The influence of the environment on the fluorescence behavior of indicator molecules is investigated. A model is developed to describe the fluorescence decay of indicator molecules in a non-uniform medium. Its consequences for fluorescence decay time based chemical sensors are discussed and verified in two examples, namely a pH sensor using a pyrene compound in hydrogel and a ruthenium complex for oxygen sensing embedded in a polystyrene membrane.

Fluorescence decay studies of photoexcited porphyrin-triptycene-quinones as biomimetic model systems for the primary processes of photosynthesis have been performed. These dyes were chosen on the basis of their rigid structure, high electron transfer (ET) rates and their advantageous absorption properties. The extremely short fluorescence lifetimes caused by an intramolecular ET process favor their use as standards in ps-lifetime measurements. Depending on the meso-substitution and metallation of the porphyrin, as well as the reduction potential of the quinone, different lifetimes in the range of 1 to 83 ps are obtained. The syntheses, spectroscopic properties and the application of these dyes will be described.

Our work is devoted to the analysis if exponential fluorescence decays, convoluted with instrumental function of a device. We suggest methods of deconvolution and estimation of exponential parameters, based on the transformation of the convolution integral to linear differential equation with constant coefficients. Integrating this equation and applying least squares method (for smoothing experimental data), one can obtain the set of linear equations for parameters. These methods can be adopted for analysis of anisotropy curves, global analysis of the curves, registered at the number of wavelengths, temperatures or concentration of solution, reconstruction of true decay via the intensity curve of reference compound radiation. Simulation model of the process of optical radiation registration was designed. It consists in the numeric generation of the measured intensities and additionally two sources of distortion: random (Poisson and Gaussian) noise and nonrandom (background, shifts). The accuracy of estimation is determined by z² criterion and residuals autocorrelation function. The standard deviation for parameters can be calculated by bootstrap method or analytically. The investigation of the deconvolution methods let us work out some recommendations for the optimal usage of the obtained methods in the processing of experimental data.

Using subnanosecond laser spectrofluorometry, the spectral and polarization time-resolved characteristics of 1-phenylnaphthylamine (1-AN) fluorescent probe in phospholipid bilayer and red blood cell membranes have been studied. It is shown that the electronic spectra of probe in model membranes are inhomogeneously broadened. In contrast to low-molecular weight solvents there are two reasons for inhomogeneous broadening. The first is connected with different levels of location of probe molecules in the membrane. Inhomogeneous broadening due to the first factor has a static character. The second reason is similar to that of solutions and linked with fluctuations of solvate structure. This type of broadening has a dynamic character. The process of intermolecular relaxation in membranes is accompanied by the release of the free energy excess, which results in wavelength-dependent rotation of probe.

In this study we report on the interfacial kinetic behavior of the widely used laser dye Rhodamine 6G in two different matrices frequently used for dye immobilization in fluorescence sensing applications. The water content of Nafion membranes and Silica Gel has been shown to be the critical parameter which determines the probe motion. Fluorescence anisotropy measurements indicate a free rotation of the R6G in wet and dry Nafion even through this cationic dye is held electrostatically within the water pores of the system. These results indicate that in Nafion the interfacial microviscosity of the water pore increases as water is removed but the dye position does not changes. Conversely in the case of silica gel the R6G is initially hindered in its motion when the water content is high but becomes free to rotate as the gel is dehydrated. This indicates that the level of water within the sol gel pore determines the strength of the interaction between the charged surface and the rhodamine 6G.

This study compares two formats of rapid assays for the detection of pesticides (bromacil and pyrethroid based metabolites): enzyme linked immunosorbent assay (ELISA) and immunoassay with near-infrared (NIR) fluorescence detection. NIR dye immunoassay (NIRDIA) measurements were carried out by using two different instruments, both having a silicon photodiode as the detector and a laser diode for excitation. ELISA and NIRDIA were performed in a tracer format, where the specific antibody is bound to the surface of a microtiter plate well and the tracer with enzyme or fluorescent dye label competes with the analyte for the antibody binding site. It was demonstrated that the NIRDIA is at least as sensitive as the ELISA. Both assays detect pesticides in the (mu) g/L (ppb) range. Hapten- macromolecule-NIR dye-conjugates have been synthesized with various biopolymers (e.g., proteins) as carriers. The use of carrier macromolecules enables convenient purification of the cyanine dye derivatives. The mild conjugation method of the dye is based on isothiocyanate chemistry.

Several groups have shown that laser-induced fluorescence spectroscopy can detect dysplastic changes in human colon tissues. We present an approach based on analysis of the underlying tissue microstructure for extracting histological information from such spectral signals. The method employs fluorescence microscopy and tissue optics to model the `bulk' fluorescence collected with an optical fiber probe in a clinical setting. For both colonic normal and adenoma, we measured the intrinsic fluorescence lineshapes, the spatial distributions of the fluorophores, and optical parameters of tissue. Numerical and analytical solutions to the radiative transfer equation were then used to compute fluorescence spectra. The results of the model were in excellent agreement with clinical spectra collected during colonoscopy, using 370 nm excitation. Four factors were found to be responsible for the spectral differences between normal tissue and adenoma: fluorescence of mucosal collagen, dysplastic cell, and submucosa, and hemoglobin attenuation. Preliminary results indicate that these parameters can be extracted from individual clinical spectra by reversing the modeling procedure.

Probes using luminescent transition metal complexes for determination of contents and conditions of environment are very promising. This work deals with the investigation of spectral properties of highly luminescent Ru(II) polypyridil complexes with such ligands as 2,2'-bipyridine, 1,10-phenanthroline, 4,7-diphenyl-1, 10-phenanthroline and their substitution analogues immobilized on porous glass surface. The object under investigation possesses a number of desirable features, since it combines luminescence properties of metal-ligand complexes with mechanical and technological advantages associated with employment of porous glass matrix. Emission of Ru(II) polypyridil complexes is typically dominated by a series of lowlying metal to ligand charge-transfer excited states. Luminescence spectra reveal wide structureless band with maxima ranging from 605 nm for Ru-tris-1,10-phenanthroline to 645 nm for Ru-4,7-diphenyl-1, 10-phenanthroline. The luminescence band shape and placement are practically independent of excitation wavelength. The radiative transition from the excited states is strongly influenced by external factors, such as presence of a quencher or temperature variations. The experiments revealed nonmonotonous character of temperature dependence of luminescence intensity. While the compositions are heated, the original luminescence quantum yield downfall in a temperature range up to 110 K is reversed and there appear to be an interval (110 - 200 K for Ru-4,7-diphenyl-1, 10-phenanthroline), where quantum yield rises, reaching maximum at 200 K. Subsequent heating up to 370 K is followed by renewal of quenching. This phenomena could be explained taking into account porous matrix inhomogeneity, leading to the existence of complexes with different orientation as to the matrix surface.

Novel high efficient fluorescent chelate for Eu based on diethylenethriaminepentaacetic acid and amino (beta) -diketones is proposed for time-resolved fluorescence immunoassay. The label surpasses all known chelates for europium in major spectral and luminescent characteristics. The proposed label has number of advantages over the well-known europium chelates. The assays with new fluorescent chelate do not require enhancement solution, but the sensitivity of Eu determination is the same as for DELFIA enhancement solution. The assay with new fluorescent chelate is insensitive to contamination of solutions and samples with ions of heavy metals, because the concentration of fluorescent chelate is measured and high excess of Eu prevents dissociation of fluorescent chelate complex. Techniques have been developed for covalent labeling of proteins with the new fluorescent chelate. The labelling proteins can be stored in the lyophilized state or in stabilized solution rather long and retain their immunological properties. Application of the new fluorescent chelate enables the washing step to be avoided and to develop the express non-separation assay.

Nile red non-covalently binds to Human Serum Albumin in at least two binding sites with distinctly different accessibilities for acrylamide quenching. Here, we report on the fluorescence characteristics of the probe-protein complex in various environments using both steady state and time-resolved single proton counting techniques. In particular, fluorescence depolarization measurements demonstrate that the unfolding of a protein by heat is fundamentally different from that using denaturant, regarding the changes in diffusional rotation of the probe at intermediate stages. We also exploit the fluorescence of the probe- protein complex in AOT reverse micelles, to increase our understanding of the nature of compartmentalized biological molecules. The large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size, to be observed. Moreover, comparison of acrylamide quenching of the tryptophanyl residue and bound Nile red in reverse micelles of varying composition, show that there is an induced stability in the nanosecond motions of the protein in reverse micelles of waterpool diameter 80 angstroms. Both far and near UV circular dichroism show that at this waterpool size, the protein structure is nearest it's native state. This waterpool size is about the same size as the central cavity in the molecular chaperone GroEL, which suggests that compartmentalization of proteins `in vivo' aids the protein folding process by inducing stability in the appropriate conformation as well as preventing protein aggregation.

The modified electro-optical absorption and emission methods are described as well as their use for measurements of electrical dipole moments of five aminophthalimides in ground and excited states. It is found that there exists a principal difference between properties of 3- aminophthalimide (3AP) and 4-amino-N-methylphthalimide (4ANMP) in different solvents. The equilibrated dipole moment of 3AP in its excited state is practically independent on the solvent polarity, in comparison with 4ANMP. The possible mechanism of this effect is discussed.

A miniature (1 mm diameter) all-optical photoacoustic probe for generating and detecting ultrasonic thermoelastic waves in biological media at the tip of an optical fiber has been developed. The probe provides a compact and convenient means of performing pulsed photoacoustic spectroscopy for the characterization of biological tissue. The device is based upon a transparent Fabry Perot polymer film ultrasound sensor mounted directly over the end of a multimode optical fiber. The optical fiber is used to deliver nanosecond laser pulses to the tissue producing thermoelastic waves which are then detected by the sensor. Detection sensitivities of 53 mv/MPa and a 10 kPa acoustic noise floor have been demonstrated giving excellent signal to noise ratios in a strong liquid absorber. Lower, but clearly detectable, signals in post mortem human aorta have also been observed. The performance and small physical size of the device suggest that it has the potential to perform remote in situ photoacoustic measurements in tissue.

We constructed a fiber-optic long-term monitoring system for mouse embryo, which developed a new quantitative and non-invasive evaluation method for human embryo. We monitored the changes of light scattering from mouse embryo which might relate to the state of blastomere and change in intercellular microstructures. We employed a couple of optical fibers to measure the scattering. These fibers were arranged in a light angle to the optical axis of a microscope to obtain adaptability for various measurements. A He-Ne laser with 632.8 nm in wavelength was used as a light source. The emission fiber (56 micrometers (Phi) ; core) and sensing fiber (400 micrometers (Phi) ; core, NA equals 0.43) were used. We observed the light scattering changes from the embryo, which were over 200 times higher than the noise level with 30 s in period. This result might be attributed to the structural and/or functional changes in the mouse embryo.

The current methods used for typing blood involve an agglutination reaction which results from the association of specific antibodies with antigens present on the erythrocyte cell surface. While this method is effective, it requires involved laboratory procedures to detect the cell surface antigens. As an alternative technique, uv/vis spectroscopy has been investigated as a novel way to characterize and differentiate the blood types. Typing with this technique is based on spectral differences which appear throughout portions of both the ultraviolet and visible range. The origin of these spectral differences is unknown and presently under investigation. They may be due to intrinsic absorption differences at the molecular level, and/or they may be due to scattering differences brought about by either subtle variation in cell surface characteristics, cell shape or state of aggregation. As the background optical density in these samples is identified and accounted for, the spectral differences become more defined. This work and the continuation of this project will be included in a general database encompassing a wide range of blood samples. In addition, long term goals involve the investigation of diseased blood with the potential of providing a more rapid diagnosis for blood borne pathogens.

Cryptosporidium and Giardia are enteric protozoa which cause waterborne diseases. To date, the detection of these organisms in water has relied upon microscopic immunofluorescent assay technology which uses antibodies directed against the cyst and oocyst forms of the protozoa. In this paper, the uv/vis extinction spectra of aqueous dispersions of Cryptosporidium and Giardia have been studied to investigate the potential use of light scattering-spectral deconvolution techniques as a rapid method for the identification and quantification of protozoa in water. Examination of purified samples of Cryptosporidium and Giardia suggests that spectral features apparent in the short wavelength region of the uv/vis spectra contain information that may be species specific for each protozoa. The spectral characteristics, as well as the particle size analysis, determined from the same spectra, allow for the quantitative classification, identification, and possibly, the assessment of the viability of the protozoa. To further increase the sensitivity of this technique, specific antibodies direction against these organisms, labelled with FITC and rhodamine are being used. It is demonstrated that uv/vis spectroscopy provides an alternative method for the characterization of Giardia and Cryptosporidium. The simplicity and reproducibility of uv/vis spectroscopy measurements makes this technique ideally suited for the development of on-line instrumentation for the rapid detection of microorganisms in water supplies.

This paper is a reflection by the authors on our more than twenty years of work to realize an in-vitro blood gas system based on fiber optic chemical sensors. We offer reflection based on our experiences in these projects. It is not our intention to present data for critical review but rather to ask the critical questions of the technology and its application. These are the critical questions that should be asked of every new technology up front, before numerous development projects, man years of effort and a fraction of a billion dollars are expended. These are the critical questions which would have detected the on set of the "hobby horse" syndrome. The developers of this technology fell in love with the technology. It was this love and our passions which ran un-checked and have yet only delivered one commercially successful product.

The development and improvement of oxygen sensors is continuously matter of research because it is important to quantify oxygen concentrations in biological systems. The presented hybrid fiberoptical sensor combines the common advantages of fiber optic sensors e.g. flexible connection to the place of interest and potential separation between place of measurement and measuring device with the special advantage of the optical analysis that does not consume the analyte oxygen. The O₂ sensing was tested by using (1) solutions with the dissolved oxygen indicator dye (tris(1,10-phenantroline) ruthenium (II) chloride hexahydrate), and (2) and O₂ flux optode. The O₂ flux optode consist of an oxygen permeable membrane (test membrane) combined with oxygen optodes to measure the pO₂ differences across the membrane. The O₂ flux can be calculated from the pO₂ gradient across the test membranes and its permeability. The developed measuring systems `FLOX' detects the phase shift between the sinusoidally modulated excitation light and the resulting fluorescence of the indicator. The phase shift is caused by the fluorescence lifetime that depends on the oxygen concentration. Many investigations have shown that measurements of lifetime are preferable to measurements of intensity because of higher stability. The FLOX system uses a blue light emitting diode (LED, (lambda) _peak equals 450 nm) as light source together with optical filters and a photo multiplier tube as detector. The coupling between the place of measurement and the FLOX is performed by a bifurcated fiber bundle in which single multimode fibers are statistically mixed. Its measuring end could be either immersed directly in an aqueous solution of the fluorophore or provided with a special sensor head for the measurement of the oxygen flux into a surface, for example into the surface of the human skin. First measurements demonstrated the applicability of the FLOX system to measure the O₂ flux into human skin; for example an about 20 - 50% increase of the O₂ flux after stop of the circulation could be clearly shown.

A multi-analyte sensor has been prepared that allows O₂, CO₂ and pH to be monitored simultaneously with rapid response to all three analytes. The sensor is fabricated to photodepositing analyte-sensitive polymer matrices on the distal surface of an imaging fiber. The array shows sensitivity to pH in the range 5.0 - 8.0, CO₂ from 0 - 15% and O₂ from 0 - 100%.

Continuous intra-arterial blood gas monitoring is a potentially valuable tool in the surgical and intensive care arenas. Patient oxygenation and acid base status can change rapidly and without warning. The ability to monitor pH_a, P_aCO₂ and P_aO₂ in arterial blood will be a major medical advance for the anesthesiologist and intensivist. Intra-arterial blood gas sensors are typically placed in radial arteries. In certain patient populations accurate monitoring is not possible in radial arteries due to arterial environmental factors such as hypotension, vasoconstriction and atherosclerotic disease. These same factors can make radial cannulation difficult resulting in traumatic catheter insertion, thereby further compromising flow conditions. In situations where radial artery flow is expected to be compromised, selecting a large vessel for sensor placement is desirable. We report an initial feasibility study of our blood gas monitoring system using the femoral artery as the sensing site. Clinical results are presented as well as potential advantages and disadvantages associated with monitoring in the femoral artery.

We describe a bedside arterial blood gas (ABG) monitoring system which uses fluorescent optical sensors in the measurement of blood pH, PCO₂ and PO₂. The Point-of-Care Arterial Blood Gas Monitoring System consists of the SensiCath^TM optical sensor unit manufactured by Optical Sensors Incorporated and the Tram^TM Critical Care Monitoring System with ABG Module manufactured by Marquette Electronics Incorporated. Current blood gas measurement techniques require a blood sample to be removed from the patient and transported to an electrochemical analyzer for analysis. The ABG system does not require removal of blood from the patient or transport of the sample. The sensor is added to the patient's existing arterial line. ABG measurements are made by drawing a small blood sample from the arterial line in sufficient quantity to ensure an undiluted sample at the sensor. Measurements of pH, PCO₂ and PO₂ are made within 60 seconds. The blood is then returned to the patient, the line flushed and results appear on the bedside monitor. The ABG system offers several advantages over traditional electrochemical analyzers. Since the arterial line remains closed during the blood sampling procedure the patient's risk of infection is reduced and the caregiver's exposure to blood is eliminated. The single-use, disposable sensor can be measure 100 blood samples over 72 hours after a single two-point calibration. Quality Assurance checks are also available and provide the caregiver the ability to assess system performance even after the sensor is patient attached. The ABG module integrates with an existing bedside monitoring system. This allows ABG results to appear on the same display as ECG, respiration, blood pressure, cardiac output, SpO₂, and other clinical information. The small module takes up little space in the crowded intensive care unit. Performance studies compare the ABG system with an electrochemical blood gas analyzer. Study results demonstrated accurate and precise blood gas measurement of 100 samples and 72 hour performance without need for re-calibration.

Fiber optic-based pH sensors have many advantages over the traditional pH electrodes. They are robust, self-calibrating and show negligible drift. In addition, they are capable of detecting pH changes within 0.001 pH units. These features make fiber optic pH sensor technology particularly attractive for biomedical and on-line process applications. Typically, pH sensors consist of an indicator dye immobilized in a polymer matrix. The pH of the solution is related to the relative fraction of the protonated and dissociated species of the dye, whose absorption spectrum changes according to the pH of the surroundings. The response of the pH sensor is determined by the dissociation equilibrium of the immobilized dye, its interaction with the polymer matrix and the ionic strength of the medium. In order to have meaningful pH measurements it is important to understand the sensing mechanism and the resulting absorption spectra. In this paper a model is reported that takes into consideration the chemical reaction and the osmotic and electrochemical effects resulting from the dye-membrane-ionic strength interactions. Ionic strength effects are quantified through the pKa of the immobilized dye and it is demonstrated that highly reliable quantitative pH measurements, in the range required for blood pH analysis, are feasible.

A sensor capable of simultaneous imaging and pH measurements has been prepared by coating the distal tip of a single imaging fiber with a pH sensitive material. The coated fiber is fabricated using photochemical polymerization with a spin coating technique and results in a fairly uniform coating of polyHEMA/fluorescein on the order of 5 micrometers thick. Performance data and imaging capabilities, as well as instrumentation requirements and deposition chemistry, will be discussed. Progress towards the fabrication of enzyme based biosensors utilizing this technique will also be presented.

The development of pH-based fiber optic biosensors and their uses in clinical and biotechnological applications are described. Based on a pH-sensitive optode, different biosensors for urea, penicillin, glucose and creatinine were developed. A multichannel modular fluorimeter was used to measure signals from up to three optodes simultaneously. The pH value and the buffer capacity are critical factors for biosensors based on pH probes and influence the biosensor signal. A flow injection analysis (FIA) system is used to eliminate the latter influences. With this integrated system, samples can be analyzed sequentially by the injection of a defined volume of each sample into a continuously flowing buffer stream that transports the samples to the sensors. The complex signal is transformed and analyzed by a computer system. Characteristic features of the FIA peak give information about the buffer capacity in the solution. With the help of intelligent computing (neural networks) it is possible to recognize these features and relate them to the respective buffer capacity to obtain more accurate values. Various applications of these biosensors are discussed. The pH optode is also used to monitor enzymatic reactions in non aqueous solvents. In this case the production of acetic acid can be detected on line.

A penicillin biosensor has been fabricated by photodepositing penicillin-sensitive polymer matrices and pH-sensitive polymer matrices on different regions of an optical imaging fiber. Penicillin is detected by coupling the enzymatic activity of penicillinase with the pH sensitivity of fluorescein. Penicillin concentration is correlated to the pH change in the microenvironment of the penicillin-sensitive matrix relative to the pH of the sample solution. This dual sensor removes the need to maintain a constant solution pH when measuring penicillin and should enhance greatly the application of biosensors.

An integrated optic refractometer device was developed to perform a rapid one-step, label-free immunoassay. The device measures refractive index changes at the surface of a planar waveguide using interferometry. Antibodies were applied to the waveguide surface to provide a bioselective coating for detecting and quantifying a specific antigen of interest. The detection limit of this biosensor was determined for adenovirus as a model for other viral analytes of military, medical, and environmental interest. As binding of the antigen occurred on the sensor surface, a time-dependent phase shift of the helium-neon laser light beam was detected and was measured over a 10-minute time period. Adenovirus was detected at levels of 250 - 2500 viral particles/ml. This detection limit was obtained for a mono-layer of antibody attached to the sensor. Use of a high-density, multi-layer antibody coating approach resulted in improved detection limits for bacteria and protein analytes of general interest.

A thin, organic film has been applied to the sensing area of a surface plasmon resonance sensor to provide specific sites for the covalent immobilization of proteins. Previously, protein binding experiments were carried out on the bare gold surface of this probe, or using a gold- thiol coupling technique. There has been great interest in increasing the probe sensitivity to protein binding reactions as well as maximizing the amount of protein immobilized in the sensor area. A new surface chemistry has been especially developed to meet these goals. This thin (approximately 200 angstroms) film is comprised primarily of a poly(ethylene oxide) (PEO)-like layer which provides a non-fouling base that resists non-specific protein uptake. Amine groups are distributed throughout this base, providing sites to which specific proteins can be immobilized using established chemistries. The two film components are deposited simultaneously by a radio frequency plasma deposition reaction. Film surface chemistry was studied by electron spectroscopy for chemical analysis (ESCA). By derivatizing the surface layer with pentafluorobenzaldehyde, using ESCA, we could relate surface fluorine signal to amine content. Data will be presented on the immobilization of biotin and the dynamic reaction of the immobilized biotin with streptavidin.

Here we wish to present automated laser spectrofluorimeter and some applications of it. The excitation part of the spectrofluorimeter includes an atmospheric pressure nitrogen laser (pulse duration 0.35 ns, peak power 350 kW) and distributed-feedback dye laser (tuning range 400 - 750 nm, spectral width 0.1 - 0.6 nm). It is possible to excite the probe by nitrogen laser or dye laser. The signal of fluorescence is registered by boxcarintegrator and data are input into the computer IBM PC/AT-286. Laser spectrofluorimeter is full automated, lasers, all optical and electronic systems are controlled by the computer. The laser spectrofluorimeter can measure time-resolved fluorescence characteristics with temporal resolution from subnanoseconds to milliseconds: (1) instantaneous fluorescence spectra (0.2 ns - 10 ms), (2) instantaneous anisotropy spectra and anisotropy kinetics (0.2 ns - 10 ms), (3) fluorescence kinetics and lifetime of excited state (0.05 ns - 10 ms). Experimental data can be treated with a full set of programs: (1) deconvolution of fluorescence pulse with up to 3 exponential approximation, (2) calculation of anisotropy kinetics with simultaneous deconvolution for different polarization, (3) calculation of instantaneous spectra from fluorescence decays, (4) mathematical treating of spectra (position of spectrum, halfwidth, asymmetry etc.).

Microchannel plate photomultipliers (MCP-PMTs) are commonly used for single photon counting fluorescence decay measurements. Recent developments to improve the pulse height distribution are described. This enables the tube to be better matched to amplifiers, constant fraction discriminators and the ensuing electronics. Multi-anode tubes are also produced for multi-channel spectroscopy. Recent measurements of crosstalk in such tubes are presented and discussed.

General aspects of immunological analyses are reported and the application of some lanthanide complexes in fluoroimmunoassays is discussed. The photophysical properties of the complexes of cage-type ligands, which, up to now, showed the most intense metal luminescence are reported. The luminescence intensity of these complexes is discussed considering the efficiency of the incident light/emitted light conversion, defined as the product of the absorption efficiency of the ligand and the metal luminescence quantum yield upon excitation in the ligand. It is illustrated how the luminescence intensity can be enhanced by adapting the ligands on basis of the previously obtained results.

In the development of a fiber optic Raman scattering, selective absorbent-based anesthetic sensor, chemometric analysis of the spectroscopic data must include a method for correction of both multiplicative and additive effects. Multiplicative signal correction (MSC) has been used effectively in near-infrared reflectance measurements to account for differing pathlengths. MSC performs a least-squares regression of each spectrum on a reference spectrum; it was successfully applied to both simulated and experimental Raman spectra. Raman spectra were obtained from dilute mixtures of the anesthetics halothane, isoflurane, and enflurane in perfluoropolyether oil. The C-H stretch peaks of the anesthetics were used by partial least squares (PLS) to distinguish between the anesthetics and predict their concentrations. The broad C-F band from the perfluoropolyether oil was used by MSC to approximate the multiplicative and additive coefficients for correction of the spectra. The MSC-corrected data yielded significantly better predicted residual error sum of squares values in PLS than the uncorrected data. These encouraging results show that MSC may be generally used in correcting for multiplicative and additive effects in spectra that include a peak from a substance that exists in approximately the same concentration throughout the work.

In numerous cases the technique of Fiberoptic Evanescent Wave Spectroscopy (FEWS) offers great advantages over regular IR spectroscopic methods. It provides an easy way for measuring the absorption spectra of highly absorbing or highly scattering samples. With FEWS one can perform measurements in situ and in real time and this is potentially useful for measurements on biological samples or for the monitoring of chemical reactions. In the present work, the FEWS technique was used to analyze human blood serum using a Fourier Transform Infrared Spectrometer. A special cell based on IR transmitting non-toxic silver halide fibers was designed. Further improvements in the analysis were obtained by adopting some multivariate calibration techniques that have already been used in clinical chemistry. The blood constituents analyzed were: urea, total protein, cholesterol, uric acid and calcium. Good agreement between our results and the ordinary chemical and enzymatic methods was obtained.

The temperature dependence of the luminescence lifetime is mainly used for temperature monitoring. But for luminescence quenching based chemical sensors it presents the major disturbance. We have investigated the temperature dependence of the luminescence decay times of the Ru[4.7 Ph₂phen]₃ ²⁺-complex, used as oxygen concentration monitor within the temperature range between 15 and 80 degree(s)C. The primal decay times (tau) ₀ without quencher and (tau) with quencher are temperature dependent. The sensitivity of the sensor, expressed on the base of the STERN-VOLMER coefficient K_SV is not temperature dependent.

We have developed a method to analyze quantitatively the biochemical composition of human coronary artery in situ using near infrared Raman spectroscopy. Human coronary arteries were obtained from explanted hearts after heart transplantation. Samples of normal intima/media, adventitia, non-calcified and calcified plaque were illuminated with 830 nm light from a CW Ti:Sapphire laser. The Raman scattered light was collected and coupled into a 1/4 meter spectrometer that dispersed the light onto a liquid nitrogen cooled, deep-depletion CCD detector. Raman spectra with sufficiently high S/N for extracting biochemical information could be collected in under one second. The spectra were analyzed using a recently developed model to quantitate the relative weight fractions of cholesterol, cholesterol esters, triacylglycerol, phospholipids, protein, and calcium salts. After spectral examination, the artery samples were biochemically assayed to determine the total lipid weight and the amount of the major lipid categories as a percentage of the total lipid content. The results of the lipid biochemical assay and the Raman spectral model compare favorably, indicating that relative lipid weights can be accurately determined in situ. Protein and calcium salts assays are underway. This in situ biochemical information may be useful in diagnosing atherosclerosis and studying disease progression.