This paper reports a bespoke adder-based deep learning network for time-domain fluorescence lifetime imaging (FLIM). By leveraging the l1-norm extraction method, we propose a 1-D Fluorescence Lifetime AdderNet (FLAN) without multiplication-based convolutions to reduce the computational complexity. Further, we compressed fluorescence decays in temporal dimension using a log-scale merging technique to discard redundant temporal information derived as log-scaling FLAN (FLAN+LS). FLAN+LS achieves 0.11 and 0.23 compression ratios compared with FLAN and a conventional 1-D convolutional neural network (1-D CNN) while maintaining high accuracy in retrieving lifetimes. We extensively evaluated FLAN and FLAN+LS using synthetic and real data. A traditional fitting method and other non-fitting, high-accuracy algorithms were compared with our networks for synthetic data. Our networks attained a minor reconstruction error in different photon-count scenarios. For real data, we used fluorescent beads' data acquired by a confocal microscope to validate the effectiveness of real fluorophores, and our networks can differentiate beads with different lifetimes. Additionally, we implemented the network architecture on a field-programmable gate array (FPGA) with a post-quantization technique to shorten the bit-width, thereby improving computing efficiency. FLAN+LS on hardware achieves the highest computing efficiency compared to 1-D CNN and FLAN. We also discussed the applicability of our network and hardware architecture for other time-resolved biomedical applications using photon-efficient, time-resolved sensors.
Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology.
BACKGROUND: Specific and sensitive nucleic acid (NA) testing in research and clinical diagnostics is usually performed by use of labeled oligonucleotide probes. However, the use of target-specific fluorogenic probes increases the cost of analysis. Therefore, universal sequence-dependent (USD) NA detection methods have been developed to facilitate cost-effective target detection using standardized reagents. CONTENT: We provide a comprehensive review of the current methods for fluorescence-based USD NA detection. Initially, we focus on the emergence of these methods as a means to overcome the shortcomings of common NA detection methods, such as hydrolysis probes and molecular beacons. Thereafter, we provide a critical evaluation of the individual detection methods. These methods include (a) target amplification with bipartite primers introducing a universal detection tag to the amplicon (UniPrimer PCR, universal fluorescence energy transfer probe PCR, attached universal duplex probe PCR, and universal strand displacement amplification) or combined with bipartite probes comprising a universal detection region (mediator probe PCR, universal strand displacement amplification, universal quenching probe PCR) and (b) amplification-independent assays employing either a universal variant of the invader assay or universal NA hybridization sensors. We discuss differences between the methods and review clinical applications. SUMMARY: The current methods for USD NA testing are cost-effective and flexible and have concordant analytical performance in comparison with common probe-based techniques. They can detect any target sequence by the simple use of a label-free, low-cost primer or probe combined with a universal fluorogenic reporter. The methods differ in the number of target specificities, capability of multiplexing, and incubation requirements (isothermal/thermocycling). Extensive clinical applications comprise detection of single-nucleotide polymorphisms, study of gene expression, in situ PCR, and quantification of pathogen load.
The article presents the history of the development and the current state of the apparatus for the detection of interferents and biological warfare simulants in the air with the laser-induced fluorescence (LIF) method. The LIF method is the most sensitive spectroscopic method and also enables the measurement of single particles of biological aerosols and their concentration in the air. The overview covers both the on-site measuring instruments and remote methods. The spectral characteristics of the biological agents, steady-state spectra, excitation-emission matrices, and their fluorescence lifetimes are presented. In addition to the literature, we also present our own detection systems for military applications.
We have measured the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) as its fluorescence lifetime is decreased by oxygen quenching. Such studies were done on DPH dissolved in the isotropic solvent mineral oil and for DPH embedded in phospholipid vesicles of either dimyristoyl-l-α-phosphatidylcholine (DMPC) or dioleoyl-l-α-phosphatidylcholine (DOPC), each at several temperatures. In order to obtain adequate quenching increased pressures of oxygen had to be used. Oxygen quenching resulted in significant changes in intensity and anisotropy, and these effects were reversible. To control for possible effects of pressure on the systems under study, equivalent experiments were performed with nitrogen, argon, or helium forming the gas phase. Under these last-mentioned conditions, changes in intensity and anisotropy were insignificant when compared with those observed with oxygen quenching. The depolarizing rotations of the fluorophore are described by its rotation rate (R) in radians/seconds and its limiting anisotropy at times which are long compared with the fluorescence lifetime, r(∞). This latter parameter provides a measure of the degree to which the fluorophore’s environment hinders its rotational diffusion. Oxygen quenching of fluorescence provides a means to vary the fluorescence lifetime simultaneous observation of the steady-state fluorescence anisotropy allows quantitation of both R and r(∞). For DPH in mineral oil at two different temperatures we found that the values of R obtained from this quenching –anisotropy measurement agreed precisely with those obtained from steady-state anisotropy measurements and with the values obtained from differential polarized phase fluorometry (Lakowicz, J. R., et al. (1979) Biochemistry 18 (preceding paper in this issue)). Additionally, r(∞) was found to be zero. These results indicate that in mineral oil DPH behaves as an ideal unhindered isotropic rotator. In contrast. DPH embedded in lipid bilayer vesicles of DMPC behaves as an isotropic but highly hindered rotator below the phase transition temperature, as is indicated by r(∞) ≃ 0.33. Above the phase transition temperature the depolarizing rotations become significantly less hindered, r(∞) ≃ 0.03. In DOPC vesicles the depolarizing rotations are unhindered at all temperatures. The temperature profiles of R and r(∞) obtained for DPH in lipid bilayers were in agreement with those observed using differential polarized phase fluorometry. Quenching–anisotropy measurements of the type we have described provide a powerful method for investigation of time-resolved decays of fluorescence anisotropy without the direct use of time-resolved methods. The estimation of membrane microviscosity from steady-state anisotropy measurements assumes that the nature of the depolarizing rotations of the fluorophore in the membrane are identical with those in an isotropic reference solvent. Our results indicate that this assumption is invalid. We estimated the apparent membrane viscosity by three methods: (1) from steady-state anisotropy measurements; (2) from the rotational rate of DPH within its hindered environment; and (3) from the diffusivity of molecular oxygen. Each method yielded a different value with steady-state polarization giving the highest and oxygen diffusivity the lowest. These results show that any quantitative estimate of microviscosity depends critically upon the molecular process used for its estimation.
Abstract Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1–9. In experiments with fluorescein‐labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady‐state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH‐sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor‐acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion‐dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor‐acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 m M and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low (5 m M ) and high buffer (1 M ) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state pK a . The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK a and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion‐dianion reaction does not significantly occur at 5 m M phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pK a * of the excited monoanion‐dianion proton reaction is around 6.3. The results and methods presented here should be useful in the development and testing of pH‐sensitive labeling fluorophores and fluorescent indicators.
Molecular imprinting technology (MIT), often described as a method of making a molecular lock to match a molecular key, is a technique for the creation of molecularly imprinted polymers (MIPs) with tailor-made binding sites complementary to the template molecules in shape, size and functional groups. Owing to their unique features of structure predictability, recognition specificity and application universality, MIPs have found a wide range of applications in various fields. Herein, we propose to comprehensively review the recent advances in molecular imprinting including versatile perspectives and applications, concerning novel preparation technologies and strategies of MIT, and highlight the applications of MIPs. The fundamentals of MIPs involving essential elements, preparation procedures and characterization methods are briefly outlined. Smart MIT for MIPs is especially highlighted including ingenious MIT (surface imprinting, nanoimprinting, etc.), special strategies of MIT (dummy imprinting, segment imprinting, etc.) and stimuli-responsive MIT (single/dual/multi-responsive technology). By virtue of smart MIT, new formatted MIPs gain popularity for versatile applications, including sample pretreatment/chromatographic separation (solid phase extraction, monolithic column chromatography, etc.) and chemical/biological sensing (electrochemical sensing, fluorescence sensing, etc.). Finally, we propose the remaining challenges and future perspectives to accelerate the development of MIT, and to utilize it for further developing versatile MIPs with a wide range of applications (650 references).
Abstract A neural network‐based method is developed to assess the vertical distribution of (1) chlorophyll a concentration ([Chl]) and (2) phytoplankton community size indices (i.e., microphytoplankton, nanophytoplankton, and picophytoplankton) from in situ vertical profiles of chlorophyll fluorescence. This method (FLAVOR for Fluorescence to Algal communities Vertical distribution in the Oceanic Realm) uses as input only the shape of the fluorescence profile associated with its acquisition date and geo‐location. The neural network is trained and validated using a large database including 896 concomitant in situ vertical profiles of High‐Performance Liquid Chromatography (HPLC) pigments and fluorescence. These profiles were collected during 22 oceanographic cruises representative of the global ocean in terms of trophic and oceanographic conditions, making our method applicable to most oceanic waters. FLAVOR is validated with respect to the retrieval of both [Chl] and phytoplankton size indices using an independent in situ data set and appears to be relatively robust spatially and temporally. To illustrate the potential of the method, we applied it to in situ measurements of the BATS (Bermuda Atlantic Time Series Study) site and produce monthly climatologies of [Chl] and associated phytoplankton size indices. The resulting climatologies appear very promising compared to climatologies based on available in situ HPLC data. With the increasing availability of spatially and temporally well‐resolved data sets of chlorophyll fluorescence, one possible global‐scale application of FLAVOR could be to develop 3‐D and even 4‐D climatologies of [Chl] and associated composition of phytoplankton communities. The Matlab and R codes of the proposed algorithm are provided as supporting information.
BACKGROUND: We developed gradient HPLC methods for quantification of the antimitotic drug irinotecan (CPT-11) and its four metabolites, SN-38, SN-38 G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin (APC), and 7-ethyl-10-[4amino-1-piperidino]-carbonyloxycamptothecin (NPC), as the sum of the lactone and carboxylate forms, in human plasma and saliva. Camptothecin was used as internal standard. METHODS: The sample pretreatment involved protein precipitation with methanol-acetonitrile (50:50 by volume) followed by acidification with hydrochloric acid to convert the lactone ring-opened form into its lactone form, quantitatively. HPLC separation was performed on a Xterra RP18 column. The excitation wavelength was 370 nm, and the emission wavelength was set at 470 nm for the first 24 min and then at 534 nm for the next 4 min. The stabilities of irinotecan and its four metabolites in plasma, saliva, and acidic extracts were also investigated under various conditions. RESULTS: Assays were linear in the tested range of 0.5-1000 micro g/L. For the five analytes, limits of quantification were 0.5 micro g/L in both matrices. The interassay imprecision (as relative standard deviation) was 3.2-14% in plasma and 2.6-5.6% in saliva. Assay recoveries ranged from 92.8% to 111.2% for plasma and 100.1% to 104.1% for saliva. Mean extraction recovery from plasma or saliva was 90%. CONCLUSION: The developed assay can be used to determine pharmacokinetic parameters for CPT-11, SN-38, SN-38 G, APC, and NPC in plasma and saliva from patients with metastatic colorectal cancer.
Reactive oxygen species (ROS) have been found in plants, mammals, and natural environmental processes. The presence of ROS in mammals has been linked to the development of severe diseases, such as diabetes, cancer, tumors, and several neurodegenerative conditions. The most common ROS involved in human health are superoxide (O2•−), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH). Organic and inorganic molecules have been integrated with various methods to detect and monitor ROS for understanding the effect of their presence and concentration on diseases caused by oxidative stress. Among several techniques, fluorescence and electrochemical methods have been recently developed and employed for the detection of ROS. This literature review intends to critically discuss the development of these techniques to date, as well as their application for in vitro and in vivo ROS detection regarding free-radical-related diseases. Moreover, important insights into and further steps for using fluorescence and electrochemical methods in the detection of ROS are presented.
Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.
BACKGROUND AND OBJECTIVE: In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of "flat" urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5-aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: Several hours after bladder instillation of an aqueous solution of ALA in 34 patients, a Krypton ion laser or a filtered Xenon arc-lamp was used to excite PpIX fluorescence. Tissue samples for histological analysis were taken while observing the bladder wall either by means of a video camera, or by direct endoscopic observation. RESULTS: A good correlation was found between the PpIX fluorescence and the histopathological diagnosis. On a total of 215 biopsies, 143 in fluorescent and 72 in nonfluorescent areas, all visible tumors on white light cytoscopy appeared in a bright red fluorescence with the photodetection technique. In addition, this method permitted to discover 47 unsuspected carcinomatous lesions on white light observation, among which 40% were carcinoma in situ. CONCLUSION: PpIX fluorescence induced by instillation into the bladder of 5-ALA is an efficient method of mapping the mucosa in bladder carcinoma.
Fluorescence methods are commonly used in pharmaceutical drug discovery to assay the binding of drug-like compounds to signaling proteins and other bio-particles. For binding studies of non-fluorescent compounds, a competitive format may be used in which the binding of the compound results in displacement of another fluorescently labeled ligand. Highly-sensitive measurements within nano-liter sized open probe volumes can be accomplished using a confocal epi-illumination geometry and thus key tools for such drug-binding studies include fluorescence correlation spectroscopy (FCS) and its related techniques. This paper reviews the general protocol for application of FCS to biomolecular compound-binding assays and it focuses on methods for the reduction of experimental photon count data to obtain the normalized autocorrelation function (ACF), on theoretical models of the ACF, and on statistical and systematic errors in the experimental ACF. Results from a detailed Monte Carlo simulation of FCS, which are useful for testing theoretical models and validating short-duration assay capabilities, are discussed. An illustrative example is presented on the use of FCS to assay binding of Alexa-488-labeled Bak peptide with Bcl-x(L), which is an intracellular protein that acts to protect against programmed cell death.
Fluorescence labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide was shown to be a promising approach toward the accurate determination of carbonyls in cellulosic materials. Combined with gel permeation chromatography in DMAc/LiCl with fluorescence/multiple-angle laser light scattering/refractive index detection, the method yields carbonyl profiles relative to the molecular weight of the cellulosic material. The derivatization procedure can be carried out either homogeneously in DMAc/LiCl or advantageously as heterogeneous derivatization in aqueous buffer. The heterogeneous carbonyl group determination, offering shorter reaction times and increased simplicity as compared to the homogeneous approach, was comprehensively validated. The carbonyl content in numerous dissolving pulps of different provenience has been determined, including pulps with carbonyl contents additionally increased by oxidative treatment. The method was also applied to follow bleaching sequences and oxidative treatments of pulps.
PURPOSE: This paper reports a technique that enables the quantitative determination of the concentration of gold nanoparticles (GNPs) through the accurate detection of their fluorescence radiation in the diagnostic x-ray spectrum. METHODS: Experimentally, x-ray fluorescence spectra of 1.9 and 15 nm GNP solutions are measured using an x-ray spectrometer, individually and within chicken breast tissue samples. An optimal combination of excitation and emission filters is determined to segregate the fluorescence spectra at 66.99 and 68.80 keV from the background scattering. A roadmap method is developed that subtracts the scattered radiation (acquired before the insertion of GNP solutions) from the signal radiation acquired after the GNP solutions are inserted. RESULTS: The methods effectively minimize the background scattering in the spectrum measurements, showing linear relationships between GNP solutions from 0.1% to 10% weight concentration and from 0.1% to 1.0% weight concentration inside a chicken breast tissue sample. CONCLUSIONS: The investigation demonstrated the potential of imaging gold nanoparticles quantitatively in vivo for in-tissue studies, but future studies will be needed to investigate the ability to apply this method to clinical applications.
The development of specific fluorescently labeled cell surface markers have opened the possibility of specific and quantitative noninvasive diagnosis of tissue changes. We are developing a fluorescence scanning imaging system that can perform a "noninvasive optical biopsy" of the Sjogren syndrome (SS) which may replace the currently used histological biopsy. The diagnosis of SS is based on the quantification of the number of topical preadministered fluorescent antibodies which specifically bind to the lymphocytes infiltrating the minor salivary glands. We intend to scan the lower lip, and for each position of the scan, generate a two-dimensional (2-D) image of fluorescence using a charge-coupled device (CCD) camera. We have shown previously that our diffuse fluorescent photon migration theory predicts adequately the positions and strengths of one and two fluorescent targets embedded at different depths in tissue-like phantoms. An inverse reconstruction algorithm based on our theoretical findings has been written in C/sup ++/ and uses 2-D images to predict the strength and location of embedded fluorophores. However, due to large numbers of variables, which include the optical properties of the tissue at the excitation and emission wavelengths, and the positions and strengths of an unknown number of fluorophore targets, the validity of the final result depends on assumptions (such as the number of targets) and the input values for the optical parameters. Our results show that the number of fluorophore targets reconstructed for each scan is limited to two, and at least the scattering coefficient at the excitation wavelength is needed a priori to obtain good results. The latter can be obtained by measurements of spatially resolved diffuse reflectance at the excitation wavelength that provides the product of the absorption and scattering coefficients.
We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.
We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.
A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.
Olive Oil -Constituents, Quality, Health Properties and Bioconversions 64 techniques, relying on recording of single emission or excitation spectra, are often insufficient if directly applied. In such cases, total luminescence or synchronous scanning fluorescence techniques are used, improving the analytic potential of the fluorescence measurements. With contributions from numerous analytes, the autofluorescence of olive oil exhibits numerous overlapping bands. Such complex spectra should be analyzed using multivariate and multiway methods.