We describe a system that records simultaneously the temporal profile of both linear polarization components of all wavelengths in an emission spectrum. Our excitation source is the vacuum ultraviolet storage ring of the National Synchrotron Light Source at Brookhaven National Laboratory, which provides a continuous spectrum of ultraviolet, visible and near-infrared light consisting of approximately equals 1 ns FWHM pulses at a repetition rage of approximately equals 50 MHz, and with identical temporal profiles at all wavelengths, although any source with similar temporal properties could be used. A single excitation band is selected by a monochromator and linearly polarized before reaching the sample. Fluorescence can be monitored either along an axis perpendicular to the excitation beam, or at near normal incidence. A polarizer divides the fluorescence into components with polarizations parallel and perpendicular to the polarization of the incident beam. The emission spectrum is dispersed by an imaging spectrograph, and detected with a resistive-anode imaging photomultiplier operated in a single photon counting mode. The time of arrival of a photon is derived from signals originating in the micro-channel plates that function as the `dynodes' of the photomultiplier, while the location of the centroid of the electron cascade on the anode of the detector indicates both the wavelength and polarization of the detected photon. Simultaneous acquisition of the time-resolved emission spectra for both polarization components is more efficient than conventional approaches and reduces the complications in data analysis that can arise when the properties of a sample change during the time when sequential data-sets are collected.

A novel method for real-time imaging of fluorescence lifetimes employing a new framing camera will be introduced. Based on a unique operating principle, this framing camera can be gated with a 480 picosecond shutter. The camera consists of an image converter with a pair of deflection electrodes and an aperture. As the photoelectron image passes through the deflection electrodes, an electric field is applied externally. The photoelectron image is thus deflected, and swept on the aperture. High speed gating can hence be accomplished. We are now constructing a fluorescence lifetime imaging microscope system employing this framing camera whereupon the repetition rate has been increased to 4 MHz. Although application of this method requires multiple acquisitions at different delay times, it enables us for the first time to observe real-time intracellular phenomena. This is achieved by combing the system with a dual-view assembly, composed of a pair of beam splitters and mirrors which produce a certain delay, hence enabling us to capture two time-resolved images simultaneously in a single operation of the framing camera. These two images have different delay times, each of which can be set from 300 picoseconds to 2 nanoseconds. The two images are used for the analysis based on the assumption that the decay is single exponential. We will describe an example of application of the system towards the observation of cellular phenomena.

We report the demonstration of a high temporal resolution fluorescence lifetime imaging (FLIM) system using a time- gated image intensifier to provide whole field FLIM images. The gate width has been optimized to 110 ps, and changes in the environment of a fluorescent phantom, causing lifetime differences of 20 ps, have been detected. Environmental changes of the fluorescent indicator, Lucifer Yellow, have been sensed by measuring changes in its fluorescence lifetime when unbound and when bound to the protein albumin.

Recent FDA-approval of topotecan (9-dimethylaminomethyl-10- hydroxycamptothecin) and camptosar (CPT-11) along with the accelerated clinical development of related camptothecin drugs provides new hope for the successful treatment of human cancer, including neoplasms for which no effective treatments currently exist. Current clinical efforts worldwide are aimed at optimizing the therapeutic efficacies of the camptothecins, with the major focus on the determination of the most effective dosing schedules. To this end, technological advances which provide a direct and rapid means of measuring plasma drug levels (i.e. such that correlations between plasma drug levels and clinical responses can be sought) would be of great utility. Here we report on the direct fluorescence detection of topotecan and SN-38 in human plasma and topotecan in whole blood at micro molar levels using two-photon excitation at 730 or 820 nm. Topotecan was detected at concentrations as low as 0.05 and 1 (mu) M in plasma and whole blood, respectively. Since skin, blood and other tissues are translucent at long wavelengths, our results suggest the attractive possibility of homogeneous or noninvasive clinical sensing of camptothecins in situ using two-photon excitation.

We demonstrate the application of three photon excitation to fluorescence probe studies using time-correlated single- photon counting. By exciting with 120 fs Ti:sapphire laser pulses at 800 nm we have observed fluorescence emission from the scintillators p-Terphenyl (PT) and 2,1 Naphthyl, 5- Phenyloxazole ((alpha) -NPO). For solutions of (alpha) -NPO in cyclohexane and PT in propylene glycol the laser power dependence of the fluorescence is consistent with the emission being due to three-photon excitation of the same emitting S₁ state which is populated with one-photon excitation at 267 nm. However, for (alpha) -NPO in alcohols some evidence for a mixture of three and two photon excitation is observed. This solute dependence correlates with the red edge of the one-photon absorption spectra. The observation of excimer emission and fluorescence anisotropy of (alpha) -NPO in small unilamellar vesicles (SUVs) of L- (alpha) -dipalmitoylphosphatidylcholine excited at 800 nm provides a clear illustration of the potential for using three photon excitation in fluorescence probe studies of microheterogeneous media. In SUVs the time-resolved data is consistent with a heterogeneous distribution of (alpha) -NPO molecules between isolated sites and ground state clusters in a similar manner to that which we reported previously for 2,5-diphenyloxazole.

A new improved method of fluorescence correlation spectroscopy, fluorescence correlation spectroscopy with traveling interference fringe excitation, for determining motional states of particles is described. In this method, the modulated fluorescence signal from particles excited by an interference fringe moving at constant velocity is detected. The autocorrelation function of the fluorescence intensity consists of terms which are characterized by the size of the excitation region and the spacing of the fringe. A lock-in detection method is adopted for extracting cosine and sine Fourier coefficients at the frequency of the traveling fringe (F_cos(t), F_sin(t)). Autocorrelation functions of F_cos(t) and F_sin(t) were found to express the motion of the fluorescent particles. The potential of this method for analyzing of the motional modes of particles in biological membranes is discussed.

By combining near-infrared (NIR) fluorophores and commercially available laser diodes, a promising technique emerges where visible probes are less effective due to background interference. The application of NIR fluorophores in fiber-optic probes for the determination of metal ions in the environment and for biological assays will be discussed. The spectral behavior of a new NIR fluorophore TG-170 in the presence of metal ions and the first synthesis and spectral characterization of a NIR dye KVA-22 substituted with a crown ether, a metal complexing functionality, will be presented.

Fluorescence-based fiber-optic sensors are the subject of considerable interest because of their high sensitivity and excellent specificity and versatility. Currently, most fluorescence sensors use UV or visible excitation and are based on intensity measurements; i.e. the emission intensity of the fluorescent indicator changes in response to binding of analyte or to some other variable of interest. While intensity-based sensors may be easy to implement in a controlled laboratory environment they are often difficult in real-world situations because of photobleaching, sample turbidity, fluorophore washout, and fouling of optical surfaces. Another drawback of intensity-based sensing is the problem of referencing the intensity measurements. A promising approach to obviate many of these problems is fluorescence lifetime sensing. Fluorescence lifetimes are exquisitely sensitive to the microenvironment of the fluorophore. Additionally, lifetime measurements are robust, being highly immune to photobleaching, and to changes in fluorophore concentration, exciting light intensity, optical density of the sample, detector sensitivity, fouling of optical surfaces, etc. Despite these advantages, the complexity, size, and high cost of currently available instrumentation for measuring fluorescence lifetimes presents a major barrier to practical implementation of lifetime-based sensors. In this paper we describe a compact, simple-to-use, inexpensive phase fluorometer suitable for sensor applications.

The non-iterative Prony's method is discussed for the deconvolution of multi-exponential fluorescence decays. The performance of these algorithms in the case of a two- exponential decay process is evaluated, using a Monte-Carlo simulation, in terms of the estimation errors caused by the signal noise. The results which are presented show that the performance of Prony's method can be greatly improved with the selection of an optimized observation window width and a few algorithm-related parameters. Comparison between Prony's method and the Marquardt least-squared-error algorithm is also made, showing the performance of the former is close to that of the latter with a 98% reduction in calculation running time. The applications of Prony's algorithms in real-time, quasi-distributed temperature sensor systems are discussed and the experimental results are presented to justify the use of the algorithms in Prony's method in practical double exponential fluorescence decay analysis.

Laser wave-mixing spectroscopy is presented as a simple, sensitive, on-column detection method for capillary electrophoresis. The use of a single focusing lens to focus and mix two input beams significantly simplifies the optical alignment requirement of this nonlinear laser method. High signal collection efficiency allows excellent detection sensitivity for both fluorescing and non-fluorescing analytes, since the signal is a sharp coherent beam. This laser detection method can be conveniently interfaced to capillary-based separation systems since it offers small detector probe volumes, efficient use of short excitation or absorption path lengths, efficient use of low laser power levels available from small, compact, inexpensive laser sources, and inherently narrower peak widths (i.e., squared Gaussian). Potential applications include sensitive detection of biomolecules, either labeled or in their native forms, using appropriate excitation wavelengths. Furthermore, laser wave-mixing detection can be used also in the indirect detection mode where the solvent or the buffer system yields a baseline signal, and non-absorbing analytes can be measured indirectly in the form of negative peaks.

The complex temporal evolution of exocytotic release of serotonin and proteins from individual rat peritoneal mast cells was monitored. Laser-induced native fluorescence with 275- and 305-nm excitation was used to detect the Polymyxin-B sulfate (Pmx) stimulated exocytosis in capillary electrophoresis (CE) and imaging microscopy, respectively. Events are observed that are consistent with released serotonin from single granules (250 aL each). With CE, a detection limit of 1.7 amol (S/N equals 3; rms) was obtained for serotonin. Following the injection of a cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with sub-second resolution. The average amount of serotonin observed per cell was 1.6 +/- 0.6 fmol; the average percentage of serotonin released was 28 +/- 14%. In complementary experiments, exocytosis was monitored temporally and spatially using native fluorescence imagery microscopy. Real time chemical images of serotonin and protein released from individual cells are obtained. The images show that the amount of material released and the time delay of the event varied from cell to cell, and that different regions of a cell behave asynchronously in releasing material.

We have been exploiting high density oligonucleotide arrays to carry out sequence analysis of genetic material from diverse sources. The method utilizes the hybridization of fluorophore labelled nucleic acids to the array and interpretation of the resulting spatial pattern of fluorescence. Our ability to obtain sequence information from the array is governed by the interplay of the synthesis and hybridization chemistry, the photophysics of the fluorophores and background interferences, and the performance of the fluorescence imaging system. The high photolithographic resolution and large usable area of the synthesis process and the presence of submonolayer coverages of fluorophores dictate that the fluorescence detection system meet several potentially conflicting performance criteria. High spatial resolution, high sensitivity, large field of view, low chromaticity and image distortion, and high dynamic range are required simultaneously. Suitable nucleic acid-fluorophore conjugates should have high absorption cross sections and emission quantum yields, low photobleaching quantum yields, and resistance to transient saturation under intense illumination. Our approaches to the design and photophysical characterization of the detection process will be discussed within the context of improving the volume of sequence information and detection limits.

In this study, we had two objectives: (1) to develop an algorithm based on the Laguerre expansion of kernels technique for deconvoluting time-resolved fluorescence spectra; (2) to characterize the time-resolved emission of elastin and collagen, substances present in different amounts in healthy and diseased arterial wall. The transient fluorescence of purified samples excited with a Nitrogen laser pulse (3 ns) was measured at different wavelengths with an MCP-PMT and digitized at 2 Gsample/s. The deconvolution algorithm expressed the impulse response function as a weighted sum of Laguerre functions. We found that five Laguerre functions were sufficient to represent the fluorescence impulse response function for both substances. A fast-decay and a slow-decay components were identified in the impulse response function. The slow decay increased with the wavelength of emission ((lambda) ) for elastin (p < 0.05) whereas it decreased with (lambda) for collagen. The fast decay was independent of (lambda) for elastin and decreased with (lambda) for collagen. The fluorescence impulse response function can be retrieved even when the duration of the excitation pulse is in the range of the fluorescence lifetime. The dynamics of the temporal emission of collagen and elastin varies with the wavelength of emission suggesting that using temporally-resolved fluorescence spectra would improve optical analysis of the arterial wall.

These studies represent further investigations that have been done utilizing the fluorescence from pyridinoline, one of the major crosslinks of type I and III collagen, to evaluate cervical connective tissue changes during various female reproductive periods. Based on our previous studies, a prototype instrument has been constructed. The instrument was specifically designed for the purpose of vaginal examination of cervical connective tissue by measuring light induced fluorescence directly from the surface of the external os of the cervix. The studies were carried out on nonpregnant rats, rats during gestation at different periods, rats at different times during postpartum, and rats during preterm birth after being treated with antiprogesterone drugs. A study has also been done on humans during pregnancy and postpartum. The results parallel previous investigations that have used various invasive methods to analyze cervical extensibility, cervical collagen content and collagenase. In consideration of the important role of the collagen fibers and their turnover in the process of cervical function during pregnancy (softening or ripening at term), this method could be a useful tool for evaluating treatment strategies of the cervix. Moreover, the instrument could serve as a device for the non-invasive estimation of cervical status in the clinic and the diagnosis of the changes in the cervix during the preparation for labor.

The fluorometric properties of indocyanine green has been largely described. Salts, proteins, lipoproteins are mentioned as modifying the fluorescence characteristics of ICG. We have recently observed that ICG is able to be bound at the interface of model membranes. In this study, we reinvestigated the spectral properties of ICG in biological media. The fluorescence quenching curves of ICG in the water, protein solution and whole blood are very similar but the absorption characteristics of ICG are quite different from one medium to another, ICG displays an aggregative behavior in water and serum depending on its concentration but we observed no modification of the absorption spectra in blood. This quenching property is also observed in vivo using blood sampling. These results show that the spectral behavior of ICG in biological media may be taken in account when fluorescence measurements are performed.

The feasibility of employing fluorescent agents to perform optical imaging in tissues and other scattering media has been examined through experimental and computational studies. Fluorescence lifetime and yield can give crucial information about local metabolite concentration or environmental conditions within tissues. This information can be employed towards disease detection, diagnosis, and treatment if non-invasively quantitated from re-emitted optical signals. However, the inverse problem for image reconstruction of fluorescence yield and lifetime is complicated due to the highly scattering nature of the tissue. In this work, a light propagation model employing the diffusion equation is used to account for the scattering of both the excitation and fluorescent light. Simulated measurements of frequency-domain parameters of fluorescent modulated AC amplitude and phase-lag are used as inputs to an inverse image reconstruction algorithm which employs the diffusion model to predict frequency-domain measurements resulting from a modulated input at the phantom periphery. In the inverse image reconstruction algorithm, we employ a Newton-Raphson technique combined with Marquardt algorithm to converge upon the fluorescent properties within the medium. The successful reconstruction of both the fluorescence yield and lifetime in the case of heterogeneous fluorophore distribution within a scattering medium has been demonstrated without a priori information or without the necessity of obtaining `absence' images.

Each year, more people at younger ages are diagnosed with primary brain tumors. Current histological discrimination between normal and diseased tissue occurs after tissue excision. A reliable optical biopsy for open craniotomy would optimize the amount and types of tissue removal by making an accurate evaluation before excision. The presented work is part of a study investigating the clinical diagnostic potential of Raman spectroscopy for gliomas. It has been shown that the optical properties of in vitro tissue are strongly dependent upon sample preparation. The investigation of the effects of time latency, paraformalin tissue fixation, and tissue perfusion with carbogen-bubbled cortical transport solution on their respective Raman spectra of brain tissue and tumors will be discussed, as well as their implications on the study of neurological tissue. The studies are conducted with in situ tissue samples from scid mice and 785 nm pulsed alexandrite laser excitation. Results illustrating positive qualitative and quantitative variations between Raman spectra of normal and malignant brain tissue will be presented.

In continuing efforts to develop a Raman `optical biopsy' technique, the lipid and protein distribution in an animal model system was differentiated without the use of invasive contrast agents. Tissue component discrimination is based on the unique vibrational spectra intrinsic to lipids and proteins. Component discrimination is possible using ratiometric image analysis techniques. However, chemical image contrast is enhanced when processing techniques are employed that use more the Raman spectrum than simply one or two Raman spectral bands. Contrast enhancement is quantitated by using an edge detection technique to objectively evaluate the effectiveness of multivariate processing.

Confocal Raman spectral imaging is discussed in terms of its application to studies within living cells. The main difficulty which arises, keeping in mind that cell viability should be respected, is the low sensitivity of Raman spectroscopy. We describe here some possible solutions to this problem: optimization of the instrumental conditions as well as use of surface-enhanced Raman scattering (SERS). SERS imaging is dependent on topology of the metal surface which is necessary to observe this effect. The advantages and limitations of different types of the SERS-active substrates when used for confocal Raman imaging of cells are analyzed using examples. We describe here the procedures and precautions we took when investigating metal-cell interfaces. The confocal Raman-SERS images presented demonstrate the applicability of SERS technique to selective analysis of intracellular distribution and molecular interactions of antitumor drugs.

This paper presents a preliminary application of Raman spectroscopy in conjunction with the chemometric method of Partial Least Squares to predict silicone concentrations in homogeneous turbid samples. The chemometric technique is applied to Raman spectra to develop an empirical, linear model relating sample spectra to polydimethysiloxane (silicone) concentration. This is done using a training set of samples having optical properties and known concentrations representative of those unknown samples to be predicted. Partial Least Squares, performed via cross- validation, was able to predict silicone concentrations in good agreement with true values. The detection limit obtained for this preliminary investigation is on par with that of magnetic resonance spectroscopy. The data acquisition time for this Raman based method is 200 seconds, which compares favorably with the 17 hour acquisition required for magnetic resonance spectroscopy to obtain a similar sensitivity. The combination of Raman spectroscopy and chemometrices shows promise as a tool for quantification of silicone concentrations from turbid samples.

A simple, low cost technique is described for producing miniaturized fiber optic chemical sensors, and results obtained using these devices to monitor pH within the micro- chemical environment of individual mammalian cells (e.g. mouse embryonic fibroblasts) is reported. The technology is based on the use of submicron optical fiber tips which have fluorescent chemical or biological reagents immobilized on their tip surface. Fiber tips (0.1 - 1 micrometers ) were formed by drawing single-mode optical fiber (125 micrometers outer diameter) in a commercial fusion splicer. Fluorescent dye-doped sol- gel films and polymers were then deposited on the surface of the fiber tip using dip-coating techniques and photochemical synthesis respectively. Laser light from an argon ion laser was coupled into the fibers untapered end, and the fluorescence light equipped with a cooled photomultiplier detection system. The sensors that have been developed using these coating technologies are reversible and have response times of a few tenths of a second. We are currently expanding this sensing technique to monitor other biomedical parameters (e.g. oxygen concentration), by utilizing alternative chemical indicators that are compatible with the immobilization techniques described.

Artificial hosts such as crown ethers, cryptands, calixarenes, and others have been used for detecting metal ions or others mostly in organic solvents. On the other hand, we prepared many cyclodextrin derivatives bearing one or two chromophores and found that they can be used as sensors for detecting various organic compounds in aqueous solution. Cyclodextrins (CDs) are spectroscopically inert, but they can be converted into spectroscopically active hosts by modification with appropriate chromophores. The modified CDs usually form self-inclusion complexes by including one or two chromophore moieties in their cavities. Fluorophore-modified CDs exhibit guest-responsive fluorescence intensity variation and the mechanism for signal transduction from guest binding to fluorescence response involves the conformational changes of modified CDs as shown by the exclusion of the fluorophore moiety from inside to outside of the CD cavity in guest binding. Here, we show molecular recognition and sensing abilities of various new types of modified CDs which bear naphthalene, dansyl, p-(dimethylamino)benzoyl or pyrene unit as a fluorescent moiety.

In comparison to metal ions in aqueous solutions, there are few methods for analysis of small anions such as cyanide, cyanate, carbonate, sulfide, and nitrate. Yet such analytes are important as environmental pollutants and as reagents and byproducts of industrial processes, paper manufacture, and mining. For some time we have been developing fluorescence-based fiber optic biosensors for metal ions such as zinc, cobalt, copper, mercury, nickel and cadmium, using the unparalleled selectivity and avidity of a metalloenzyme, human carbonic anhydrase. In the cases of Cu²⁺, CO²⁺, and Ni²⁺, we made use of the characteristic weak d-d absorbance bands of these metals when bound in the active site of the enzyme to serve as a fluorescence energy transfer acceptor for a suitably positioned fluorescent label attached to the enzyme. For this approach the intensity and lifetime of the fluorophore reflect the degree of energy transfer, and therefore the concentration of the metal. To measure certain anions such as cyanide and cyanate, we made use of the well-known perturbation of the d-d absorbance of Co²⁺ when an anion inhibitor becomes bound and inhibits the enzyme. These changes in absorbance modify the overlap integral with a suitable fluorescent label, and thereby the degree of energy transfer, resulting in a perturbation of the intensity and lifetime.

Benthic aquatic environments like biofilms or sediments are often investigation by measuring profiles of chemical or physical parameters at a high spatial resolution (< 50 micrometers ). This is necessary to understand e.g. transport processes and the biogeochemistry of the sediment water interface. A variety of electrochemical and optical microsensors has been developed and used for this purpose. In most of these applications the temperature of the investigated biofilms or sediments is assumed to be constant. However measurements with thermocouples of an appr. diameter of 300 micrometers have shown that this is not always the case for illuminated shallow water sediments and biofilms. We developed new microoptodes for measuring temperature distributions at a high spatial (< 50 micrometers ) and thermal (< 0.2 degree(s)C) resolution in aquatic systems. The new sensors are based on a fluorophore that is well known for its application in oxygen sensing-Ruthenium(II)- tris-1,10-phenantroline. Demas et al. (1992) discussed the possible use of highly luminescent transition metal complexes as temperature indicators. We have approached this idea from our experiences with ruthenium complexes as oxygen indicators. The first realized sensor consists of a closed microcapillary filled with an indicator solution and in inserted tapered optical fiber. The principle uses the temperature dependence of the fluorescence lifetime in the solution. To keep the solution oxygen free an oxygen scavenger is added to it. The change of the lifetime is detected by a special measuring device that uses a phase modulation technique.

Based on the recently introduced T-Sensor method, we demonstrate the fluorescence-determination of various analytes directly in whole blood and in serum. The method relies on microfluidic flow in silicon structures, diffusion-based separation, and analyte determination using fluorescent and absorption indicator dyes. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without mixing other than by diffusion. A sample solution (e.g., blood), and a receptor solution containing the indicator dye are introduced in a common channel, and flow laminarly next to each other until they exit the structure. Small ions such as H⁺, and Na⁺ diffuse rapidly across the channel, whereas larger molecules diffuse more slowly. Larger particles such as blood cells and polymer beads show no significant diffusion within the time the two flow streams are in contact. The fluorescence emission of indicator dyes is a function of the concentration of the analyte molecules and the dye concentration in the interaction zone between the two streams. This device allows continuous monitoring of the concentration of analytes in whole blood without the use of membranes or prior removal of blood cells. This principle is illustrated by the determination of human albumin, total calcium, and pH in whole blood and serum.

A free path surface fluorosensor laser system, capable of giving a real time indication of the presence of phytoplankton at or near the sea surface, is reported. The system utilizes an Argon ion laser operating at 488 nm, the output beam of which is amplitude modulated, expanded and directed onto the water surface. The resulting phytoplankton fluorescence is collected via a simple telescope and filter arrangement and then passed to a phase sensitive detection system. Initial tests using a single phytoplankton species (Thalassiosira pseudonana) carried out in the laboratory have shown that the system is able to detect phytoplankton (500 k cells/ml) at ranges up to 15 m.

We have developed a fluorescence lifetime based energy transfer sensor for detecting copper(II) ions. Rhodamine 800 in Nafion selectively transfers its energy to copper ions when excited at 670 nm. By fitting to the fluorescence decay we can resolve copper concentrations in water down to the level of 10 ppb. Time-correlated single-photon counting is used for detection. Good discrimination against interference by cobalt, nickel and chromium ions is obtained by virtue of the reduced spectral overlap of these ions as compared to copper. Possibilities for an energy transfer sensor for both copper(I) and copper(II) ions are discussed. Application of the theory of resonance energy transfer is investigated for use in sensor matrices using a range of donors and acceptors and the appropriateness of current models discussed.

A series of near-IR fluorescent dyes have been prepared which contain an intramolecular heavy atom for altering the photophysics to produce a set of probes appropriate for single lane DNA sequencing applications. The identification of the terminal nucleotide base will be affected by temporal discrimination using fluorescence lifetime determination. The heavy-atom modification consists of an intramolecular halogen (mono- or disubstituted) situated on a remote section of the chromophore in order to minimize the perturbation on the photophysics. The series of dyes prepared showed similar absorption and emission maxima as well as fluorescence quantum yields that were similar. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution, yielding an apparent inverse heavy atom effect, with the heavier atom showing the longest fluorescence lifetime. Nanosecond flash photolysis spectroscopy of these dyes indicated that the intersystem crossing rates in the series increased with the heavier atom, consistent with known heavy-atom effects. The apparent inverse heavy atom effect resulted from decreases in the internal conversion rate of the base chromophore, with the heavier atom showing a smaller rate of internal conversion compared to that of the dye with the lighter heavy-atom modification.

We examined the spectroscopic properties of a novel class of reporters for biomedical diagnostics called upconverting phosphors. These materials exhibit high emission efficiencies, extremely low background levels, no photobleaching, minimal phototoxicity and a variety of emission bands suitable for multi-analyte assays. In addition, they are buffer insensitive and can be excited with inexpensive and compact diode lasers. These properties are very useful for immunoassays, nucleic acid assays, in vivo diagnostics, and other applications. We synthesized submicron monodisperse phosphors and measured the emission spectra, excitation spectra, and power dependencies for a variety of phosphor materials containing different active rare earth ions and host materials. In this paper, we discuss the relation between spectral properties and diagnostic applications.

Cyanine-based near-IR fluorescent probes have been applied as labels to determine aldehyde-groups in polymeric materials. The advantage of such probes is that they can be applied to polymers which are autofluorescent or strongly colored or to strongly scattering polymer emissions. The example which is discussed in this paper is the labelling of a polymeric core-shell latex, comprising a polystyrene core and a glycidylmethacrylate shell, which has been hydrolyzed and subsequently oxidized to form aldehyde functional groups, by application of a Cyanine with a hydrazide functionality. The Cyanine which has been applied absorbs and fluoresces in the 650 - 700 nm range. In addition to near-IR fluorescence, the Cyanine dyes have strong absorption (with extinction coefficients in the order of 2 X 10⁵ I/mol.cm). It is shown that by application of reflection absorption measurements also sensitive detection of labelled functional groups can be realized. The combined application of fluorescence and reflection measurements allows for the detection of concentration and distribution of functional groups in polymeric systems down to very low levels.

In the near infrared range fluorescent background signals are very small and it is possible to reach high sensitivity in the detection of labeled compounds. With phosphorescent compounds as labels, it is possible, firstly, to add microsecond temporal resolution for background rejection for NIR labels and thus to improve sensitivity. Secondly, compounds that are phosphorescent in NIR are very promising for oxygen life-time imaging of living tissue. Several different groups of palladium and zinc porphyrins and phthalocyanins (meso-tetraphenyl)-(tetrabezo)-porphyrin, meso-tetraphenyl-(tetranaphtho)-porphyrin, tetraazaporphyrins, phthalocyanines) which possess strong absorbance in NIR range were synthesized and analyzed for room temperature phosphorescent properties in organic solvents and in water solution. Among them only Pd- tetrabenzo-(tetraphenyl) porphyrins have high quantum efficiency (10%) with the life-time 328 us and excitation 630 nm, emission 800 nm. In the NIR spectral range water strongly quenches the long-lived phosphorescence of metalloporphyrins. Metalloporphyrins can form inclusion complex with cyclodextrines in which water quenching is almost eliminated. Quantum efficiency and life-time in cyclodextrin solutions are the same as in organic solvents. We analyzed the influence of three different cyclodextrines (alfa, beta and gamma) on the phosphorescent properties of Pd-porphyrins and highest enhancement of the phosphorescence signal occurred for hydroxypropilated (Beta) -cyclodextrin.

Maintaining antibody function after immobilization is critical to the performance of a biosensor. The conventional methods to immobilize antibodies onto surfaces are via covalent attachment using a crosslinker or by adsorption. Often, these methods of immobilization result in partial denaturation of the antibody and conformational changes leading to a reduced activity of the antibody. In this paper, we report on the immobilization of antibodies onto the surface of an optical fiber through an avidin-biotin bridge for the detection of ricin, ovalbumin, and Bacillus globigii (Bg). The assays are performed in a sandwich format. First, a capture antibody is immobilized, followed by the addition of the analyte. Finally, a fluorophore- labeled antibody is added for the specific detection of the analyte. The evanescent wave-induced fluorescence is coupled back through the same fiber to be detected using a photodiode. In all cases, we observe an improved performance of the biosensor, i.e., lower limit of detection and wide linear dynamic range, for the assays in which the antibody is immobilized via avidin-biotin bridges compared to covalent attachment method.

The Amplified Spontaneous Emission (ASE) from the non- rigidized and rigidized amino-coumarins C1 and C102 respectively in p-dioxane, were studied for various added quantities of water, using a picosecond laser at 355 nm for excitation. While the ASE spectrum of C102 remained unchanged, that of C1 displayed an additional long wavelength ASE band at 445 nm, along with the normal ASE at 415 nm, found in the absence of water and the relative intensities of the long wavelength ASE to the short wavelength ASE increased with increasing water content. Addition of polar solvents such as acetonitrile to p-dioxane solutions of C1 did not produce the additional longer wavelength ASE. These findings indicate that apparently the TICT excited states of C1 are involved in this long wavelength ASE with the H-bonding water molecules facilitating their stabilization. These generally non radiative TICT states of C1 apparently then complex to form bicimers, whose allowed spontaneous transitions get amplified through the gain of the medium. Apparently under conditions of weak excitation such as in conventional fluorescence studies, one does not have adequate numbers of C1 bicimers and/or gain to be able to detect their emission as a second band at long wavelengths in p-dioxane water solutions.

We have designed, fabricated and tested a compact excitation-emission spectrofluorimeter with optical fiber light delivery and collection for use in rapid analysis of tissues in a clinical setting. This system provides ten different excitation wavelengths, permitting collection of all ten emission spectra from biological tissues in approximately 500 ms. It uses a nitrogen laser which pumps a sequence of dyes placed in cuvettes on a rotating wheel. All dye cuvettes utilize a common optical resonant cavity, which simplifies the coupling of the different excitation wavelengths into the delivery fiber of an optical fiber probe. A filter wheel containing one long-pass filter for each excitation wavelength is synchronized with the dye wheel for compactness and ease of control. A white light excitation source permits acquisition of the tissue diffuse reflectance spectrum on each cycle. The fiber optic probe is composed of seven optical fibers arranged at the distal tip in the form of a central delivery fiber surrounded by six collection fibers, with an optical shield. Return fluorescence and reflected light are dispersed by a small spectrograph and detected by a photodiode array detector. The system can collect a single shot spectrum from biological tissue with a signal-to-noise ratio in excess of 50:1. This unique design for providing rapidly variable excitation wavelengths facilitates the collection of multi- excitation fluorescence spectra for the diagnosis of diseases in vivo.

One of the promising recent developments in fluorescence microscopy is fluorescence lifetime imaging microscopy. This type of microscopy images the lifetime of fluorescence molecules (in the nano second range) rather than the amount of light emitted by these molecules. This physical property is of interest while it gives information about the local environment of the molecule, such as molecular concentration of O², Ca²⁺, pH, and conjugation. Our goal is to design a affordable, robust and easy-to-use FLIM workstation which is completely automated and does not need any difficult calibration. Therefore we are developing a workstation which applies a homodyne detection scheme (frequency range: 1 - 100 MHz) with use of an intensity modulated laser-diode (635 nm) and a gain modulated intensified CCD camera to image fluorescence lifetimes in the range of 1 - 100 ns. Using these components it is possible to make a FLIM workstation based on a normal fluorescence microscope by just replacing the light source and image detector. The FLIM image acquisition procedure in software allows automatic optical measurements of fluorescence lifetimes in different ranges and mixtures of lifetimes by adjusting the modulation frequency.

A flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making frequency-domain, excited-state lifetime measurements on cells/chromosomes labeled with fluorescent probes, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence signals are processed by (1) low-pass filtering to obtain conventional FCM dc-excited signals and (2) phase-sensitive detection electronics to resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase-shifts and to quantify fluorescence lifetimes in real time. Processed signals are displayed as frequency distribution histograms and bivariate contour diagrams. Recent examples of biological applications include: (1) lifetime histograms recorded on autofluorescent human lung fibroblasts, murine thymus cells labeled with antibodies conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, and on cultured cells, nuclei, and chromosomes stained with DNA-binding fluorochromes and (2) phase-resolved, fluorescence signal- intensity histograms recorded on autofluorescent HLFs labeled with immunofluorescence markers and on murine thymus cells labeled with Red 613-antiThy 1.2 and propidium iodide (PI positive `dead' cells) to demonstrate the resolution of signals from highly overlapping emission spectra. This technology will increase the number of fluorescent markers usable in multilabeling studies and lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.

We observed fluorescence emission from p-terphenyl, 2,5- diphenyl-1,3,4-oxadiazole (PPD) and indole resulting from two-photon excitation with two different wavelengths near 380 and 760 nm. For two-color two-photon (2C2P) excitation the emission spectra and intensity decays were the same as observed with single photon excitation with an equivalent energy near 250 nm. The two-color two-photon induced emission was observed when the samples were illuminated with both wavelengths, but only when the ps laser pulses were spatially and temporally overlapped. The signals were typically 50-fold to 1000-fold less for illumination at 380 or 760 nm alone. When illuminated with both wavelengths, and when both beams were simultaneously attenuated to the same extent, the emission intensity depended quadratically on the total illumination power, indicating two-photon excitation. When the illumination intensity at one wavelength was attenuated, the signal depended linearly on the power at each wavelength, indicating the participation of one-photon at each wavelength to the excitation process. For 2C2P excitation with both beams vertically polarized the time- zero anisotropies were larger than possible for single photon excitation. For PPD and p-terphenyl with intensity depended on the polarization of each beam in a manner consistent with co-linear transitions, but more complex behavior was found for indole. These results demonstrate that two-color two-photon excitation can be readily observed with modern ps laser sources. This phenomenon can have numerous applications in the chemical and biomedical sciences, as a method for spatial localization of the measured volume.

Fluorescent compounds based on the benzothiazole ring system 1 are prepared. The effect of substituted benzothiazole derivatives on absorption maxima is investigated. Absorption and emission (lambda) _max of all compounds are recorded. The structures are characterized by NMR. These fluorescent compounds will be used as precipitable fluorophores in the development of a prototype sensor for the monitoring and analysis of antibody/antigen reactions using an optical technique.

A 12-capillary prototype electrophoresis system for DNA sequencing has been constructed. Laser illumination is introduced into an optical waveguide that is formed by an array of individual capillaries that serve both as the optical elements of the periodic array and as the channels containing sieving media for electrophoresis. A theoretical framework and experimental data will be presented to illustrate the viability of this approach.

We report on the development of a novel experimental set-up using laser atomic fluorescence for detection and concentration measurements of heavy metal atoms for environmental and biomedical analyses. This spectrometer is based on the application of tunable LiF:F₂^+** and LiF:F₂^- color center and alexandrite lasers with nonlinear converters for narrowband excitation of atomic fluorescence and the use of gated multichannel CCD detectors for fluorescence measurements. A standard graphite furnace module was used for sample atomization. The laser sources used provide narrowband selective laser excitation continuously tunable in the 200 - 400 nm range and are therefore suitable for resonant excitation of atomic transitions in practically all known heavy metal atoms. In the first experiments, water samples containing Cu, Pb and Fe impurities were studied and detection levels of less than 1 ppb were observed. Comparison of the results of atomic laser fluorescence analysis and traditional atomic absorption spectrometry showed good qualitative agreement between these two methods. It is projected that full optimization of our experimental set up will allow for improved detection levels of several orders of magnitude. Possible optimization and simplification of the spectrometer are discussed in the context of developing a portable instrument for field use.

Modified electro-optical absorption and emission methods were used to measure the dipole moments of six coumarin fluorescent probes (CU1, CU4, CU7, CU30, CU120, CU334) in the equilibrated ground, excited Franck-Condon and equilibrated excited states. The measurements were performed in cyclohexane and dioxane at room temperature. The equilibrated ground and excited states dipole measured by electro-optical methods are compared with those derived from other measurements techniques and from semiempirical calculations. Experiments and calculations performed in this work reveal a set of anomalous and interesting properties of CU7 and CU30 in solutions. The spectral dependence of some electro-optical coefficients and, consequently, dipole moments in excited Franck-Condon state indicates that the absorption band is a superposition of several (at least two) electronic transitions. Most probably there are two close- lying singlet electronic states which contribute to the first absorption band. Our electro-optical and laser spectrofluorimetry measurements do not support the formation of twisted intramolecular charge transfer state after optical excitation in the studied coumarins, at least in cyclohexane and dioxane.

We report the first read-out module for use with single- photon timing array detectors such as multi-anode MCP-PMTs. The IBH Model 5000MXR interfaces to the time-correlated single-photon counting (TCSPC) technique using a single time-to-amplitude converter. In addition to performing established multiplexing tasks, such as simultaneous acquisition of fluorescence and excitation and anisotropy, the new module enables spectral and spatial imaging of kinetic parameters such as fluorescence lifetimes and amplitudes. The system retains the inherent advantages of TCSPC with respect to picosecond time resolution and wide dynamic range, while featuring parallel data acquisition and enhanced data acquisition rates. Unlike early TTL implementations of multiplexing which were limited to four channels, our system uses an application specific integrated circuit (ASIC) which can read out the data from up to sixteen detection channels with higher reliability and less time-dispersion. The Model 5000MXR can be packaged as a NIM standard module, packaged to serve more channels or be close coupled to detector arrays for specific applications such as microscopy and lifetime based sensors. The theory, design and performance of ASIC data read-out will be described. Other applications include photon migration in tissue, time- of-flight reflectometry/mass spectrometry and nucleonics.

Algal growth on stones, together with the deposition of other soiling layers, cause major conservation problems for buildings and monuments, as it not only covers the surface with a green layer, but also accelerates stone decay. In recent years laser ablation techniques have been used to clean masonry as they are potentially less destructive than chemical or physical techniques due to the high selectivity in removing the coating covering the stone and the absence of secondary products as with conventional techniques such as use of chemicals or of sandblasting. Whilst laser ablation cleaning is finding favor in removal of surface layers from stones there has been little or no reported work relating to the effect of the laser radiation on the algae found on stones. In order to optimize any cleaning or preservation technique for algae covered stones it is necessary to have a detailed knowledge of such effects. In this paper we report some initial results from the analysis of several algae commonly found on masonry before and after irradiation at different wavelengths using two lasers, a nitrogen laser and a Nd:YAG laser.

Microspectrofluorometry and fluorescence imaging have been largely used to measure the intracellular pH of living cells. Such in vivo measurements require the knowledge of the optical properties of the tissue and the pharmacokinetic of the dye. The purpose of this study was to investigate in vitro and in vivo spectral characteristics of BCECF. In vitro, measurements in presence of blood show that blood content can greatly affect pH measurement by increasing the fluorescence ratio. In vivo, the presence of blood in high vascularized tissue leads to the modification of BCECF spectral characteristics. Fluorescence kinetic profiles provides information about tissue perfusion. Consequently, pH measurements using BCECF or fluorescein derivatives by the double excitation method may be performed taking in account the tissue blood content and the tissue pharmacokinetic of the dye.

A fiber-based wave-mixing probe is presented as a simple spectroscopic measurement tool with minimum optical alignment requirements for trace-concentration analytes. For a multi-photon nonlinear laser-induced grating method to become widely accessible, the optical setup must be compact, easy to align and portable. We demonstrate a significant improvement in the forward-scattering (self-diffraction) wave-mixing optical setup using optical fibers for both laser input and signal output interfaces. There is considerable flexibility inherent in the design, since the wave-mixing probe can be used in multiple configurations. Wave-mixing spectroscopy is presented as an effective and sensitive analytical method for trace analysis, offering advantages such as detection in very small sample volumes, remote and in-situ analyses, and convenient and efficient alignment enhancements obtained by the use of optical fibers.

Steady-state and time-resolved laser-fluorescence microscopy has been used to study the fluorescence emission from dye molecules embedded in micrometer-sized spherical polymer particles. Sharp ripple structures are observed in the fluorescence emission spectra of dyes. These structures are found to be consistent with the theoretical calculations of morphology dependent resonances (MDR's). Observed intensities of these MDR's in the fluorescence emission spectra are found to show excitation energy dependence. The MDR's in the beads do not appear to affect the radiative lifetime of the dyes.

We use time-resolved fluorescence anisotropy decay measurements to watch the complete process of phase- separation of molecularly mixed polymer blends. Fluorescent chromophores are covalently incorporated into one of the polymers in a blend, and electronic excitation transfer (EET) between these chromophores contributes to the rate of fluorescence anisotropy decay, which is measured using time- correlated single photon counting. Since the EET is highly sensitive to the chromophore distribution, this technique can reveal the single-chain structure and spatial distribution of polymer chains in the blend on the angstroms distance scale. Blend samples are studied both by rapidly quenching the phase-separating material below its glass- transition temperature and analyzing nanoscopic aggregates trapped in the sample, as well as by watching the process evolve in situ as the sample is heated above the glass transition and through the cloud-point. We find that not only the annealing temperature, but also the variable rate of heating can have a dramatic effect on the aggregation state of the blend at the nanoscopic level. Therefore, we may be able to influence those physical properties of the blend which depend on the aggregation state in specific ways. We examine the onset of phase separation while varying the molecular weight and chromophore content of one of the blend components and the blend composition.

Bis(phenanthroline)dipyridophenazineruthenium(II), [(Ru(phen)₂dppz]²⁺, exhibits unusual optical properties compared to other derivatives of [Ru(bpy)₃]²⁺. [Ru(phen)₂dppz]²⁺ does not exhibit photoluminescence in water, but does emit brightly at approximately 610 nm in the presence of nonaqueous solvents or hydrated polymers like DNA and Nafion. We have observed this `light switch' effect upon binding [Ru(phen)₂dppz]²⁺ to a protein with a hydrophobic pocket (chymotrypsin), suggesting that we may be able to develop optical probes of protein tertiary structure. We have also prepared derivatives of [Ru(phen)₂dppz]²⁺ that have ancillary ligands other than phenanthroline, and find that these molecules change color upon exposure to small molecules; the environmental parameters we have been able to assess so far include Lewis acidity, the nature of hydrated counterions, polarity, and hydrogen-based donation ability.

Sensing of anions has been investigated using the fluorescence decaytime as the information carrier. The sensing mechanism is based on the coextraction of an anion and a proton, and the presence of a fluorophore with a rather long fluorescence decaytime inside the membrane to act as a pH indicator. The relevant theory is discussed shortly. As an example a sensor for nitrate is shown, and the influence of ionic additives on the working function has been investigated.

Uncharged long-wave fluorescent probes, Nile red and its derivatives varying in lipophilicity, were used for probing hydrophobic binding sites of human serum albumin (HSA) and lipoproteins (LP) in norm and pathology. The synchro-scan fluorescence spectra (synchronous scanning of both excitation and emission wavelengths at constant (Delta) (lambda) ) of the probes were studied in HSA solutions and in whole blood plasma. The parameters of the spectra were sensitive to pH-induced conformational NyieldsB transition in HSA. In blood plasma, each of the probes displayed a two-component synchro-scan spectrum revealing two pools of the dye bound to HSA (longer wavelength) and LP (shorter wavelength). The probe distribution between LP and HSA was also sensitive to NyieldsB transition. The LP/HSA probe distribution ratio was shown to increase significantly in certain pathologies, due to either hypoalbuminemia or lowered ligand-binding capacity of HSA. Also, spectral shifts were observed in the band of albumin-bound probe. The determination of the distribution parameter may be proposed as an informative and feasible diagnostic test.

To study a possibility of interaction of two optically, but not chemically coupled samples of whole human blood the following experimental setup was used. A quartz cuvette with either nondiluted blood or saline was placed inside a glass vial. Saline diluted whole blood was poured into the vial and respiratory burst (RB) was initiated in it with phorbol ether or zymosan. Luminol-dependent chemiluminescence (LCL) was registered using liquid scintillation counter (coincident circuit off). Effect of blood placed in the cuvette upon photon emission from blood placed in the vial was evaluated. It was shown that blood of some donors consistently attenuated photon emission from the sample in which RB was induced. Blood of another group of donors enhanced photon emission from the `partner' sample. Some properties of blood taken from the cuvette after being in the contact with the sample in which RB was induced changed in comparison with the same blood that was contacting with the non-stimulated sample. Exposed blood has lost the ability to attenuate light emission from the fresh portion of blood in which RB was induced. Its own LCL in response to addition of zymoscan was different from that of the parallel sample of same blood not exposed to sample undergoing RB. These results suggest that two chemically separated but optically coupled samples of blood can interact.

During the clinical endoscopic LIF-diagnostics of human digestion organs is needed to register fluorescence spectra in situ. Taking into account intensive bloodstream and therefore strong hemoglobin absorption of laser light the registered fluorescence signal is strongly dependent on distal probe geometry. The contact optics sensor is specially designed to enhance acquisition of auto- and exogenous fluorescence spectra from tissues. The fiber optics sensor consists of cylindrical sapphire or silica body with flat proximal and spherical distal end and tapered tip. The latter one has been made of optical material. This tip has been arranged abutting against the proximal end of the cylindrical body. Such sensor design offers to collect autofluorescent light in wide angle region effectively. Moreover that offers to use sensor simultaneously as an efficient both laser radiation collector and fiber coupler to transport exciting light at the testing site and backward. The produced distal probe has been used through the biopsy channel to endoscope. Original beam splitter throws 80% of fluorescent signal toward detector and simultaneously transmits up to 85% of He-Cd laser radiation to the tissue.

Method of room temperature tryptophan phosphorescence (RTTP) has been used to study slow intramolecular equilibrium motions in membrane proteins. The conventional home-made instruments were employed for measurement of RTTP kinetic and spectral parameters. Objects of the investigation were suspensions of human erythrocyte membranes, different animal and plant cells. On rat gepathocytes it has been shown that membrane proteins in composition of subcellular structures and native cells are able to the RTTP with tens and hundreds milliseconds lifetimes. An overwhelming part of soluble proteins of cytoplasm, karyoplasm and mitochondrial matrix has not capability to RTTP with lifetimes above 1 ms. It is concluded that unlike membrane proteins soluble proteins as a rule are characterized by motions of protein structure with intensive low frequency and large amplitude, that leads to pronounced quenching of their RTTP. In the case of membrane proteins, which are capable of phosphorescence in a millisecond range, the flexibility of the chromophores environment decreases. These results indicate that RTTP method gives the unique possibility to investigate dynamical structure of membrane proteins without their preliminary isolation from cells. The data on membrane proteins intramolecular dynamics in composition of cells at the action of biological active substances in physiological concentrations--Concavalin A, nerve growth factor, epidermal growth factor, 24-epibrassinosteroid received by the phosphorescent method are presented.

The detailed analysis of the processes of electronic energy relaxation in a substance, taking place after the short optical impulses of excitation, shows that mathematical models of the detected optical intensity decay can not be expressed by a combination of elementary mathematical functions. In the present work we suggest the approach to the parameter estimation of the decay curves, obtained from the time-resolved fluorescence experiments, based on the computer simulation methods. By means of simulation elementary processes of energy relaxation can be reproduced with the highest level of the detailed elaboration. Given processes are characterized by a set of parameters which have to be estimated. For the estimation we tried several methods, which do not require calculation of the derivatives, as in widespread Marquardt algorithm, that could be difficult for the decay curves, represented by a simulation model. The main advantage of the proposed approach is that it is possible to estimate the parameters of the physical processes without knowing their functional form. The series of simulation experiments on an estimation of parameters fluorescence decay kinetics confirms high potential of the considered approach.

Lateral diffusion of cell surface proteins is commonly measured by spot fluorescence photobleaching recovery (FPR) methods where the 1/e² radius of the interrogated spot is typically 0.5 micrometers . On an 8 micrometers lymphocyte the effective spot area represents only 1/500 of the total surface. An FPR lateral diffusion measurement of a protein expressed as 50,000 copies per cell thus reflects the dynamics of only 100 molecules and this greatly limits the precision and reproducibility of FPR measurements. A new method for interferometric fringe pattern FPR permits simultaneous interrogation of the entire surface of round cells. Fringe patterns are generated interferometrically within the optical path of a conventional microscope FPR system so that spot photobleaching measurements can be performed interchangeably. Methods for interpreting recovery kinetics on round cells and for determining the fraction of mobile protein are presented. Fringe FPR data of the murine MHC Class II antigen I-A^k (wt) expressed on M12.C3.F6 cells showed fluorescence signals improved 100-fold relative to spot FPR, with corresponding improvements in S/N ratios of recovery traces. Diffusion coefficients of 2.07 +/- 0.37 and 1.79 +/- 0.97 X 10^-10 cm²sec^-1 were obtained by fringe and spot methods, respectively. The corresponding mobile fractions of I-A^k were 66.1 +/- 7.8% and 63.4 +/- 18.0%. Improved reproducibility of fringe over spot results are slightly less than signal improvements predict. There may thus be substantial variation from cell to cell in proteins dynamics and this method may permit the assessment of such variation.

A fiber-optic immunosensor was used to determine concentrations of the mycotoxin fumonisin B₁(FB₁) in both spiked and naturally contaminated corn samples. Samples were extracted with a mixture of methanol/water. Two methods were used to prepare the methanolic corn extracts before introduction to the immunosensor: (1) simple dilution of the methanolic corn extract; or (2) affinity column cleanup. The sensor displayed an IC₅₀ of 70 ng FB₁/mL when toxin was introduced in phosphate buffered saline. Simple dilution of methanolic corn extracts yielded an assay with an IC₅₀ equivalent to 25 (mu) gFB₁/g corn and a limit of detection of 3.2 (mu) g/g corn, while affinity cleanup of corn extracts yielded an assay with an IC₅₀ of 5 (mu) gFB₁/g corn and a limit of detection of 0.4 (mu) gFB₁/g corn. The difference in sensitivity between the two cleanup techniques was due to concentration of fumonisins obtained from the affinity cleanup procedure. Naturally contaminated corn samples were also analyzed after either simple dilution or affinity column cleanup. For comparison the naturally contaminated corn samples were analyzed with an HPLC method after isolation of the fumonisins with strong anion exchange (SAX) solid phase extraction cartridges. The SAX/HPLC method and the immunosensor method agreed well except when large amounts of other fumonisins (i.e. fumonisin B₂) were present. This was due in part to the cross-reactivity of the monoclonal antibody with other fumonisins. The immunosensor has the potential to screen individual corn samples for fumonisins within six minutes, and is among the fastest of the currently available FB₁ detection methods.

Both theory and experimental results show the capacity to locate the presence of a fluorescent volume incurred within a scattering medium from frequency-domain measurements of photon migration. Frequency-domain measurements consisted of monitoring the phase-shift and amplitude demodulation of fluorescent and excitation light reemitted from a tissue phantom in response to a sinusoidally modulated excitation source located 2.8 cm away from a detecting fiber positioned on the phantom periphery. Measurements of modulation phase- shift and amplitude were conducted as a 9 mm diameter cylindrical heterogeneity containing micromolar concentrations of IR-125 and DTTCI in 0.5%. Intralipid was moved within a 20 cm diameter vessel filled with 0.5 Intralipid. These measurements were investigated to evaluate the contrast offered by lifetime, fluorescent yield, and the fluorophore concentration difference between the heterogeneity and its surroundings. Our results show that the degree of contrast offered by fluorescence is always superior to that afforded by light absorption, enabling better detection of a diseased tissue with preferential fluorescent dye uptake. Contrast is also enhanced by the lifetime of the fluorescent dye, its uptake into simulated diseased volume, and the volume into which preferential uptake occurs relative to the surrounding tissues.

ICG is a tricarbocyanin fluorescent dye used in angiography. Several reports point out the advantage of ICG to fluoresce in the near IR wavelength range enabling the imaging of deep tissues. If the fluorescence characteristics of ICG in buffer or protein solutions are described in vitro, little is known concerning the physicochemical properties of ICG. This study aims to evaluate the physicochemical and fluorescence spectral characteristics of ICG in aqueous solution and in presence of micelles and liposomes. ICG exhibits a tensioactive property and when incubated with micelles and liposomes tends to be aggregated or embedded at the interface. The fluorescence is very low in the aggregated form and is very high when ICG is embedded at the interface and a shift of the emission peak toward longer wavelength is observed. Such in vitro study could contribute to a better understanding of some observed unusual properties of ICG in vivo.

In this work we studied fluorescence properties of pyrene and pyrene in microsomal membranes, adsorbed on the surface of silver island film (SIF). Pyrene adsorbed on SIF from acetonitrile possesses enhanced emission in the region 420 - 500 nm. Fluorescence of pyrene monomers (360 - 400 nm) is negligible. Microsomal membranes probed by pyrene were adsorbed on a quartz plate and silver island film surfaces. In the former case the fluorescence intensity of pyrene monomer form is greater. It was found that the effect of fluorescence enhancement significantly depends on spectral properties of rough silver surface. Heat treated (annealed) according to special technique silver island films were the most suitable for fluorescence studies. It is shown that fluorescence enhancement effect is determined by the interaction of pyrene (pi) -electron system with surface plasmons of SIF. On the base of obtained results the possibility of rough metal surfaces application to fluorescence studies of microsomal membranes is discussed.

Preliminary results of the investigation of two-photon excited fluorescence of acridine orange (AO) and ethidium bromide (EB) in complexes with DNA are presented. Spectrofluorometer based on picosecond Nd:YAG laser was used for investigations on two-photon (1064 nm, 1 mJ, 40 ps) and one-photon (532 and 355 nm) excitation. Highly purified DNA from beef spleen and AO, EB were used at the concentration of: AO- 15 (mu) M, EB- 12 (mu) M, DNA- 2-15 (mu) g/ml. The spectra of two-photon excited fluorescence of AO, EB and their complexes with DNA as well as kinetics of the intensification of dye's two-photon fluorescence during their interactions with DNA in dependence on DNA concentration were obtained. The intensity of fluorescence of AO and EB was increased maximum 2.4 and 8 times correspondingly. During the one-photon excitation corresponding values were for: AO-2.5 time and EB-10 time. The difference in the intensification of EB fluorescence connected with the difference of the change of one-photon and two-photon absorption coefficients during the interaction of the dye with DNA.

Photon bursts of single rhodamine 6G and rhodamine B molecules in aqueous solution were studied by excitation with a frequency-doubled titanium: sapphire laser. Multichannel scalar traces, fluorescence correlation functions and fluorescence decays determined by time- correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. With the setup described it is possible to distinguish single dye molecules of different kind via their characteristic fluorescence lifetimes of 1.79 +/- 0.33 ns for rhodamine B-zwitterion and 3.79 +/- 0.38 ns for rhodamine 6G.

We studied photon bursts of individual rhodamine 6G molecules in ethylene glycol flowing through a microcapillary (diameter 1 micrometers ). Excitation was provided by an argon ion laser ((lambda) equals 514.5 nm). The laser beam was coupled into a confocal microscope by a glass plate and focussed onto the sample by an oil-immersion objective (magnification 100X). Fluorescence was collected by the same objective and detected by a photomultiplier. In order to reject out-of-focus signals a pinhole (diameter 100 micrometers ) was placed in the image plane. Several filters were used to suppress laser stray light and Raman scattered photons. With the setup described sequential counting of individual molecules is possible.

Using a mode-locked titanium: sapphire laser at 700 nm for two-photon excitation we studied fluorescence bursts from individual coumarin 120 molecules in water and triacetin. Fluorescence lifetimes and multichannel scaler traces have been measured simultaneously. Due to the fact that scattered excitation light as well as Raman scattered photons can be suppressed by a short-pass filter a very low background level was achieved. To identify the fluorophore by its characteristic fluorescence lifetime the time-resolved fluorescence signals were analyzed by a maximum likelihood estimator. The obtained average fluorescence lifetimes (tau) _av equals 4.8 +/- 1.2 ns for coumarin 120 in water and (tau) _av equals 3.3 +/- 0.6 for coumarin 120 in triacetin are in good agreement with results obtained from separate measurements at higher concentrations.

We developed a simple, tiny setup with only a few optical devices for fast and sensitive identification of DNA with a short-pulse semiconductor laser operating at 20 MHz. In combination with newly synthesized fluorescent dyes (rhodamine derivatives) which exhibit high fluorescence quantum efficiencies and distinct fluorescence lifetimes at semiconductor laser excitation wavelength a sensitive detection and time-resolved identification of DNA can be achieved. At an excitation wavelength of 635 nm the fluorescence background is greatly reduced. We demonstrate the DNA identification of A- and G-terminated DNA fragments labeled at the 5'-end with the rhodamine derivatives MR 200- 1 and JA 169 during capillary gel electrophoresis. The characteristic time-resolved data are acquired by the time- correlated single-photon counting technique. Time-resolved identification analysis is realized by the maximum likelihood estimator. For prediction of the error rate (misclassification) Cramers equation in combination with a pattern recognition technique is applied. These methods deliver high reliabilities at low classification error rates for low fluorescence light level applications.

We present experimental results of single B-phycoerythrin molecule detection in a fluid flow at different sample introduction rates. A new mathematical approach is used for calculating the resulting burst size distributions. The calculations are based upon a complete physical model including absorption, fluorescence and photobleaching characteristics of the fluorophore; its diffusion; the sample stream hydrodynamics; the spatially dependent optical detection efficiency; and the excitation laser beam characteristics. Special attention is paid to the phenomenon of `molecular noise'--fluctuations in the number of overlapping crossings of molecules through the detection volume. The importance of this study and its connections to experimental applications are discussed.

The use of fluorescence probes for in-vivo diagnostics is at the forefront of medical science. To transition this technique into the clinical environment, quantitative spectral analysis and knowledge of the cellular interactions of the marker probes is vital. Furthermore, fluorescence intensity and lifetime changes, as a function of physiological environment, represents a diagnostic opportunity. A new class of polyazamacrocyclic chelates of Terbium have been identified with rich spectroscopic properties. These chelates are tissue selective, have fluorescence lifetimes on the order of milliseconds, sharply spiked emission spectra (< 15 nm FWHM), large Stokes shifts (> 280 nm), good water solubility and high quantum yields (approximately 0.6 for PCTMB). We will present our in-vitro and in-vivo spectroscopic evaluation of the chelates. In addition to the spectral investigations, results from cellular binding specificity studies using Sprague-Dawley rats with UMR 108 osteosarcomas will be presented. The potential to use the Tb(III) chelates as neoplastic tissue markers will be discussed.