Proceedings Volume 1054

Fluorescence Detection III

E. Roland Menzel
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Proceedings Volume 1054

Fluorescence Detection III

E. Roland Menzel
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Volume Details

Date Published: 17 May 1989
Contents: 1 Sessions, 27 Papers, 0 Presentations
Conference: OE/LASE '89 1989
Volume Number: 1054

Table of Contents

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Table of Contents

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Practical Applications Of Infrared Fluorescence Spectroscopy
Karen Rubelowsky, Raymond Kaminski
The analytical demands of research in electro-optics, materials science and biochemistry have stimulated great interest in the longer wavelength region of the electromagnetic spectrum. New techniques and hardware refinements now allow the of fluorescence spectroscopy beyond the traditional boundaries of the ultraviolet and visible spectrum. The authors describe a system modified for research in the region above one micron. System performance is demonstrated by spectra characterizing the solid-state laser material Nd:YAG, an organic photoconductor, and a sample of chlorophyll.
Removing The Instrument Function From Fluorescence Spectra
Andrew F. Childs
The spectrum acquired at the sample phototnultiplier tube of a fluorescence spectrophotometer is a product of the sample spectrum and the instrument function. The determination of the instrument function and its removal from the acquired spectrum is often critical to the accurate determination of the physical properties of the sample. Methods are discussed for the determination and removal of the instrument function from excitation and emission spectra. Methods considered include quantum counters and ratio circuits for excitation correction, and emission correction against calibrated excitation systems, calibrated tungsten lamps, and NBS standard quinine sulfate.
Standards For Corrected Fluorescence Spectra
Ambler Thompson, Kenneth L. Eckerle
A set of four fluorescent standards has been produced and calibrated at the National Institute of Standards and Technology (NIST) for corrected relative spectral emission over the wavelength range from 400 to 740 nm. This new Standard Reference Material (SRM) 1931 has been produced in the form of sintered mixtures of inorganic phosphors and polytetrafluorethylene (PTFE) powder for spectral response calibration of spectrofluorimeters used in biology, medicine and studies of molecular structure. They provide a means for correcting spectrofluorimeter errors due to wavelength dependencies of optics, monochromators, and detectors, thus permitting meaningful intercomparison of results obtained in different laboratories.
Determination Of Fluorescent Quantum Yields Using Pulsed-Laser Photoacoustic Calorimetry
Jeanne Rudzki Small, Jonathon J. Hutchings, Enoch W. Small
Pulsed-laser, time-resolved photoacoustic calorimetry is a technique which provides information on nonradiative channels of deactivation of molecular excited states. It is thus complementary to fluorescence techniques, and can provide information on fluorescent quantum yields. This paper describes pulsed photoacoustic calorimetry, how the data can be deconvolved to give dynamic and enthalpic information, and how this information can be used to determine fluorescent quantum yields.
Extensions Of The Method Of Moments For Deconvolution Of Experimental Data
Enoch W. Small, Louis J. Libertini, David W. Brown, et al.
The Method of Moments is one of a series of closely related transform methods which have been developed primarily for the deconvolution and analysis of fluorescence decay data. The main distinguishing feature of the Method of Moments is that it has been designed to be robust with respect to several important nonrandom errors of instrumental origin. The historical development of the method is reviewed here. Several new extensions are also described, including a statistical theory, an improved global analysis, and a method for analyzing continuous distributions of lifetimes. The new statistical theory is the first to incorporate a combined treatment of exponential depression and moment index displacement, both necessary components of the Method of Moments. In comparisons with the more commonly used least squares iterative reconvolution (LSIR) approach, it is shown that, in analyses of ideal synthetic data with random noise, the Method of Moments gives deviations in recovered parameters which are slightly greater but essentially comparable to those found by the data fitting method. Real experimental data also contain nonrandom errors. In the presence of certain such errors, decay parameters recovered by the Method of Moments will be unaffected, whereas the least squares method may yield incorrect results, unless care is taken to fit all of the data errors. An example of the improved global analysis application of the Method of Moments is shown in which two rhodamine dyes with very close lifetimes are distinguished based on spectral data. Also, the use of the distribution analysis method is illustrated with the binding of the intercalating dye ethidium bromide to DNA and nucleosome core particles. At very low ionic strength the width and location of the lifetime distribution shows a time dependence, indicating time-dependent changes in the environment of the probe. Finally, examples of Method of Moments analyses are shown for a totally different kind of data, photoacoustic waveforms. Multiexponential decays can be resolved. A new version of the Method of Moments program (which runs under either DOS or OS/2 operating systems) will soon be available on request.
Recovery Of Fluorescence Lifetime Distributions Generated By Heterogeneous Systems
Aleksander Siemiarczuk, Brian D. Wagner, William R. Ware
The Maximum Entropy Method and a new Exponential Series Method are described and tested for the recovery of underlying fluorescence lifetime distributions from digital fluorescence decay data. These two numerical techniques are applied to two heterogeneous systems, the intramolecular quenching in 1,3-di(1-pyrenyl)propane and the cupric ion quenching of pyrene in a sodium dodecyl sulfate micellar system. In the former, the short lifetime region is characterized by a broad distribution, which as the temperature is lowered, shifts dramatically to long lifetimes. In the micellar system, a lifetime distribution following the predicted Poisson distribution is recovered at each of five cupric ion concentrations, a result that supports the model of quencher ions distributed in the micelles according to Poisson statistics.
Improved Methodology For Time-Correlated Single Photon Counting
Douglas Magde, Blair F. Campbell
Injecting a small fraction of the excitation pulse directly into the photomultiplier allows one to make a simultaneous recording of the instrument response function along with every sample measurement in time-correlated single photon counting procedures. The reference path is several nanoseconds shorter than the sample path, so the two signals are distinguishable. By this innovation, one can correct for drift in either the excitation pulses or the detection system including photomultiplier and electronics.
Recent Developments And Applications Of Multidimensional Fluorescence Spectroscopy
J. B. Zung, R. L. Woodlee, M.-R. S. Fuh, et al.
Fluorescence spectroscopy is an inherently selective technique; however, single wavelength measurements can be limited for the analysis of complicated multicomponent samples or even a simple mixture with overlapping emission spectra. To overcome this limitation, multidimensional fluorescence measurements can be used to obtain additional information about the sample. The multidimensional fluorescence measurement involves the use of multiple parameters to increase the specificity of the measurement. In this laboratory, we are especially interested in multiparametric measurements such as the Excitation-Emission Spectrum. In this paper a fiber optic based multidimensional fluorometer is examined along with a portable multichannel fluorcineter in terms of sensitivity. In addition, specific applications incorporating the excitation-emission matrix are presented.
Expert System For Characterization Of Fluorescence Spectra For Environmental Applications
Khalid J. Siddiqui, DeLyle Eastwood, Russell L. Lidberg
A potentially very powerful and feasible expert system for intelligent characterization of spectra is proposed. Unlike the conventional expert systems it does not solely rely on human expert knowledge; instead, it has the capability to generate the appropriate domain dependent problem solving knowledge itself. The structure of the system is based on supervised information processing techniques and machine-based pattern recognition methods. Feature extraction techniques are used to measure appropriate spectral characteristics and then a hierarchical pattern classifier is used to characterize the spectra. This system has the potential of using different classifiers as appropriate at each of the nodes. This system was used with reasonable results on a relatively simple example of 24 ultraviolet-visible synchronous fluorescence spectra of petroleum oils (heavy crudes and No. 6 fuel oils) with Euclidean distance as a measure of dissimilarity.
HPLC Determination Of Polycyclic Aromatic Compounds By Fluorescence Detected With A Charge-Coupled Device
Rafi D. Jalkian, M. Bonner Denton
A solid-state two-dimensional charge-coupled device (CCD) is used to quantitate polycyclic aromatic compounds by native fluorescence. The mixture is separated by reverse phase high performance liquid chromatography (HPLC) and the fluorescence emission from each peak is integrated by the detector. A new technique, charge-dependent variable binning, applied to one-dimensional spectroscopy leads to outstanding dynamic range. Excellent linearity and limits of detection are obtained for several priority pollutants. Synchronous and derivative techniques can be applied to the two-dimensional fluorescence data obtained to resolve overlapping peaks. The analyzed mixture consists of fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, and perylene.
Photoluminescence Spectroscopy Of Thin Indium-Tin-Oxide Contacts On Mercuric Iodide Substrates
R. B. James, X. J. Bao, T. E. Schlesinger, et al.
Mercuric iodide (HgI2) photodetectors with sputtered indium-tin-oxide (ITO) entrance electodes were studied using low-temperature photoluminesence spectroscopy. The photoluminescence spectra obtained on each photodetector was found to differ for points beneath the ITO contact and points adjacent to it, indicating that the contact fabrication process introduces new carrier traps and radiative recombination centers within the ITO-HgI2 interfacial region. In particular, a new broad band was observed in the spectra taken from points beneath the ITO electrode. Photo-current-versus-position measurements showed that the intensity of this broad band was enhanced in regions having relatively poor photoresponse. Specimens of HgI2 with evaporated semi-transparent tin and indium films were also investigated. The spectra obtained from points beneath the Sn and In films suggest that the regions having poor photoresponse in the ITO-contacted photodetector contain either free tin or indium metal.
Surface Enhanced Fluorescence Of Basic Fuchsin At Roughened Silver Electrodes
Jeanne E. Pemberton, Steven J. McCloskey
Surface enhanced fluorescence and surface enhanced resonance Raman scattering (SERRS) of the dye basic fuchsin are observed on Ag electrode surfaces which have been electrochemically roughened with an oxidation-reduction cycle (ORC) in the presence of basic fuchsin and 0.1 M KC1. The fluorescence of the dye on the Ag surface is red shifted by ca. 26 nm relative to solution fluorescence, and as time passes, the fluorescence broadens into the blue region of the spectrum. The enhancement factor is ca. 26 ± 10 for a vertically oriented monolayer of the dye on the roughened Ag electrode. Scanning electron microscopy (SEM) indicates that unique roughness features are formed during the ORC in the presence of the dye. These features appear to be necessary for surface enhanced fluorescence.
Fluorescence Decay Kinetics Of Polyester Yellow In Solutions And In Polymers
S. Gangopadhyay, M. W. Pleil, W. L. Borst
We have studied the fluorescence decay kinetics of polyester yellow in solutions and in polymers. The fluorescence of the dye in glycerol could be fitted with a sum of three exponential decays, which corresponds to three conformers differing in the orientation of the malononitrile group with respect to the dialkylaminophenyl group. At high concentrations of the dye, a ground state dimerization was observed for the first time in non-polar solvents and in polymers. From their fluorescence decay kinetics, two different configurations were identified.
Triplet Photophysics Of Polystyrene With And Without Selected Terminating Substituents
Richard D. Burkhart, Jon A. J. Burrows, Gregory W. Haggquist
Polystyrene molecules selectively terminated with an acetophenone-like (AC) group at one end and a naphthyl group at the opposite end have been prepared and their triplet photophysics investigated. Photoexcitation of solid films of these polymers using a nitrogen laser (337 nm) yields phosphorescence bands attributable to the AC group and the naphthalene group. Using 337 nm excitation and viewing the emission at 0.1 msec after the excitation pulse, a broad phosphorescence band overlaps the AC signal. At longer delay times the typical structured AC emission emerges if the sample temperature is at 77 K but at ambient temperature the broad band persists. Apparently the AC emission is inhomogeneously broadened with an inhomogeneous band width large enough so that spectral diffusion can occur at 77 K. Luminescence decay kinetics provide information about possible modes of energy transfer from initially excited AC species to the naphthyl group.
Analytical Applications Of High-Resolution Molecular Fluorescence Spectroscopy In Low Temperature Solid Matrices
J. W. Hofstraat, W. J. M. van Zeijl, F. Smedes, et al.
High-resolution fluorescence spectroscopy may be used to obtain highly specific, vibrationally resolved spectral signatures of molecules. Two techniques are presented that both make use of low temperature, solid matrices. In Shpol'skii spectroscopy highly resolved spectra are obtained by employing n-alkanes as solvents that form neat crystalline matrices at low temperatures in which the guest molecules occupy well defined substitutional sites. Fluorescence line-narrowing spectroscopy is based on the application of selective (mostly laser-) excitation of the guest molecules. Principles and analytical applications of both techniques will be discussed. Specific attention will be paid to the determination of pyrene in bird meat by means of Shpol'skii spectroscopy and to the possibilities of applying two-dimensional fluorescence line-narrowing spectroscopy.
Synchronous Fluorescence Studies Of Anthracycline Anti-Tumor Drugs
Bruce J. Tromberg, Thomas G. Burke, James H. Doroshow, et al.
Synchronous fluorescence (SF) is used to study structurally and spectrally similar anthracycline anti-tumor drugs in biological fluids and model membranes. SF bandwidths at half maxima are typically 20-24 nm versus 75-80 nm for unresolved spectra acquired during conventional scans. This substantial band-narrowing effect permits excellent resolution between Adriamycin and an interferant, 5- iminodaunorubicin, in a two-component mixture. SF detection limits for Adriamycin are 7 x 10-7 M in serum and 1.5 x 10-7 M in buffered aqueous solution. Additional studies of structurally homologous drugs in liposomes and organic solvents indicate that SF may be useful in characterizing the influence of substituent groups on relative membrane permeation depth.
Time-Resolved Fluorescence Depolarization Study Of Lamellar To Inverted Cylindrical Micellar Phase
Kwan Hon Cheng
The orientational order and rotational dynamics of 2-(3-(diphenyl-hexatrienyl) propanoy11-3-palmitoyl-L-a-phosphatidylcholine (DPH-PC) embedded in dioleoplphosphatidylethanolamine (DOPE) were studied by time-resolved fluorescence depolarization technique. Upon increasing the temperature, the wobbling diffusion constant D⊥ of DPH-PC was found to decrease at the lamellar (Lα) to inverted cylindrical (HII) phase transition (12°C). The calculated ratio of order parameter in the La phase to that in the HII phase was close to the theoretical value of 2.0 as predicted from the change in packing symmetry. The effects of butylated hydroxytoluene, cholesterol and phosphatidylchollne on this phase transition were also examined.
Distances And Distance Distributions In A Ca[sup]2+[/sup]-Binding Protein
Robert F. Steiner, Jianqing Lan, Louise Garone, et al.
If a distribution of separations exists between a fluorescent donor group and an absorbing acceptor group, then the apparent efficiency of radiationless energy transfer will be different when monitored by measurements of static fluorescence intensity or average fluorescence decay time. This may be used to compute the parameters characterizing the distribution of separations, assuming it is Gaussian in form. Parameters computed in this way agree with those obtained by the frequency domain method for model systems. The distribution of separations has been computed for a donor group linked to Met-25 of troponin C and an acceptor group linked to Cys-98. The distribution narrows in the presence of Ca2+.
Fluorescence Studies Of Jet-Cooled Solvent Complexes Of Tryptophan
Chin Khuan Teh, Mark Sulkes, Jeffrey Sipior
Tryptophan has been complexed with polar and non-polar solvent molecules using supersonic gas expansion techniques. The ensuing complexes were studied by various laser fluorescence methods (laser scanned excitation, dispersed emission, lifetime measurement). Addition of proton donating solvents always results in the appearance of a new band somewhat blueshifted from the bare molecule conformer origin transitions, near 34960 cm-1. This solvent induced band is similar to one induced in tryptamine and probably arises from analogous interactions. Unlike the case of tryptamine, however, other solvent site interaction possibilities bring about additional redshifted solvent bands as well.
Fiber Optic-Based Immunosensors: A Progress Report
Suresh K. Bhatia, Richard B. Thompson, Lisa C. Shriver-Lake, et al.
Immobilization of functional antibodies on optical fibers at high density and configuration of optical components so as to maximize the signal/noise (S/N) are key elements in the development of fiber optic-based immunosensors. Functional antibodies were immobilized on silica substrates using thiol-terminal silanes and heterobifunctional crosslinkers. Anti-IgG antibody was routinely immobilized on glass cover slips at 0.66 ng/mm2 and on silica fibers at 0.96 ng/mm2 using this procedure. The immobilized antibody bound 0.37-to-0.55 moles IgG antigen per mole antibody. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was only 2-6% of the total antigen binding. Initial assessments of antibody immobilization and function were performed using radiolabelled proteins. In order to convert the system to fluorescence, antigens were labelled with a variety of fluorophores and optical components were tested in a variety of configurations. Problems with background fluorescence were identified stemming from fluorescence of the glass substrates, stickiness of fluorescent proteins, and fluorescence from optical components. The latter problem was addressed by testing a variety of components and configurations in a sensor testbed to determine which provided the greatest sensitivity. Using this iterative approach, we were able to identify factors which degrade sensitivity and to detect 100 pM fluorophor in a distal cuvette configuration with S/N of 14.
Time-Resolved Luminescence Imaging: Application To Latent Fingerprint Detection
K. E. Mitchell, E. R. Menzel
5-methoxyninhydrin reacts with amino acids, forming a product which further reacts with TbC13•6H20. The resultant coordination compound displays ligand-rare earth intramolecular energy transfer via which long lived Tb3+ luminescene is obtained. The luminescence is effectively excited by argon-ion laser near-UV light, which can be modulated such that time-resolved imaging with a gazeable proximity-focused microchannel plate image intensifier can be utilized. This is applied to the detection of latent fingerprints located on strongly fluorescent surfaces.
Some Fluorescence Observation On The Cancernation Tissue And The Blood Of Cancer Patients
Lee Wenchong
The fluorescent characteristic of cancerous tissue and blood of cancer patients were studied by Raman-Fluoro-Spectroscopy and fluorescence spectrophotometry in our lab. Fifteen kinds of cancer, more than 250 cases were measured.
Spectroscopic Studies Of Chemical Reactions At High Static Pressures
Malcolm Nicol, Gerald Z. Yin, Choong-shik Yoo
Unsaturated carbon compounds are unstable at high pressures with respect to denser aggregates. The aggregation reactions can be accelerated by pressure, temperature, photons, and shockwaves. For most compounds, very few details are known about the products, kinetics, and mechanisms of these reactions. We have used several types of absorption, emission and light-scattering spectroscopy to study thermal or photo reactions of cyanogen and naphthalene on the microgram scale in diamond-anvil cells at static pressures to more than 10 gigapascals (GPa). In some cases, products have been recovered for identification by gas chromatography-mass spectroscopy.
Quantitative Connections Between Resonance Raman And Electronic Emission Spectra
Jeffrey I. Zink, Kyeong-Sook Kim Shin, Londa Larson
The purpose of this paper is to show the connections between resonance Raman and electronic emission and absorption spectra. The time-dependent theory of Heller is used throughout this paper because both the physical interpretation and the mathematical calculations emphasize the connections. First, the time-dependent theory of electronic spectroscopy is introduced. Next, the time-dependent theory of resonance Raman spectroscopy is discussed and the intensities of fundamentals are examined. The theory is then applied to the spectra of Rh2(O2CCH3)4(PPh3)2 and W(CO)5(Pyridine).
Electronic Excited States Of Copper(I) Substituted-1,10-Phenanthroline And Substituted-Phosphine Mixed-Ligand Complexes
G. A. Crosby, G. R. Gamble, K. J. Jordan
A series of five title complexes was investigated spectroscopically in glasses at 77 K. Absorption and emission measurements revealed that the lowest singlet charge-transfer state moves closer in energy to both the ligand triplet and the charge-transfer triplet as the basicity of the phosphine ligands increases. The result is accompanied by a change in the relative distribution of the emissions from the latter two states. As the states become closer in energy the rate of internal conversion between singlets of disparate electronic configurations becomes slower. This is a key feature of the proposed model for this optical behavior. Consequently, intersystem crossing becomes competitive with internal conversion leading to changes in the relative yields of ligand and charge-transfer phosphorescence.
Quasi-Aromatic Heterocyclics As Laser Dyes
Theodore G. Pavlopoulos, Joseph H. Boyer
Superior laser dyes were recently discovered in the quasi-aromatic families of syn-dioxabimanes and pyrromethene-BF2 complexes. The representative new dye A-bis-(carboethoxy) methylene-syn-(methylene, methyl) bimane lased at 507 nm with 50% greater efficiency than was obtained with Coumarin 30. A similar determination showed 4,4-difluoro-1,3,5,7,8- pentamethy1-4-bora-3a,4a-diaza-s-indacene to lase at 546 nm with about 300% greater efficiency than was obtained from Coumarin 545. These quasi-aromatic systems are readily available and offer desirable properties of stability (photo, thermal, and chemical), solubility, and diminished triplet-triplet absorption in the lasing spectral region. Selected spectroscopic and lasing properties of these new laser dyes are presented.
Pressure-Induced Exciton Instability In Organic Solids Probed By Fluorescence
Atsuo Matsui, Ken-ichi Mizuno
Luminescence and absorption spectra under hydrostatic pressures in the range 0-50 kbar have been studied. The instability of free excitons occurs in tetracene, anthracene and phenanthrene crystals, while the instability of self-trapped excitons occurs in α-perylene and pyrene crystals. Experimental results lead to a conclusion that the exciton-phonon coupling constants in those materials are well expressed as a function of the intermolecular distance between nearest-neighbor, plane-parallel molecules.