Light-emitting Fluorescence Research Articles

A Polarization Isolation Method for High-Sensitivity, Low-Cost On-Chip Fluorescence Detection for Microfluidic Lab-on-a-Chip

The trend in medical equipment is toward compact and integrated low-cost medical test devices. Fluorescence-based assays are used to identify specific pathogens through the presence of dyes, but typically require specialized microscopes and narrowband optical filters to extract information. We present a novel, high-sensitivity, cost-effective, cross-polarization scheme to filter out excitation light from a fluorescent dye emission spectrum. This concept is demonstrated using an inverted microscope fitted with a halide lamp as the excitation source and an organic photo voltaic (organic photodiode) cell as the intensity detector. The excitation light is linearly polarized and used to illuminate a microfluidic device containing a 1 volume of dye dissolved in ethanol. The detector is shielded by a second polarizer, oriented orthogonally to the excitation light, thus reducing the magnitude of the detector photocurrent by about 25 dB. The signal due to fluorescence emission light, which is randomly polarized, is only attenuated by about 3 dB. As proof-of-principle, the fluorescence signal from the dyes Rhodamine 6G (emission wavelength of 570 nm) and Fluorescein (emission wavelength 514 nm) are measured in a dilution series with resulting emission signal being detected by an organic photodiode. Both dyes were detectable down to concentrations of 10 nM. This suggests that an integrated microfluidic device, with an organic photodiode and an organic light emitting excitation source and integrated polarizers, could be fabricated to realize compact and economical lab-on-a-chip devices for point-of-care diagnostics and on-site analysis.

Fabrication and characterization of OLED with Mg complex of 5-chloro-8-hydroxyquinoline as emission layer

Magnesium(II) ions were complexed with 5-chloro-8-hydroxyquinoline (L) to give an MgL 2 chelate that emitted green fluorescent light in tetrahydrofuran (THF) solvent as well as in solid state when excited by UV light. The complex was found to produce electroluminescent (EL) and thus OLEDs were fabricated and characterized based on this green light emitting material. In these devices the hole transport layer was obtained using a thin film of N, N′-diphenyl- N, N′-bis(3-methylphenyl)-1,1′-biphenyl-4,4′-diamine (TPD), LiF as hole blocking layer and tri(8-hydroxyquinoline aluminum)(Alq 3) as the electron transporting layer. The EL peak was at 539 nm with 19 nm red shift from PL peak. The EL devices fabricated had a maximum brightness of 425 cd m −2.

High-sensitivity, disposable lab-on-a-chip with thin-film organic electronics for fluorescence detection

We report a high-sensitivity, disposable lab-on-a-chip with a thin-film organic light-emitting diode (OLED) excitation source and an organic photodiode (OPD) detector for on-chip fluorescence analysis. A NPB/Alq3 thin-film green OLED with an active area of 0.1 cm(2) was used as the excitation source, while a CuPC/C(60) thin-film OPD with 0.6 cm(2) active area was used as a photodetector. A novel cost-effective, cross-polarization scheme was used to filter out excitation light from a fluorescent dye emission spectrum. The excitation light from the OLED was linearly polarized and used to illuminate a microfluidic device containing a 1 microL volume of dye dissolved in ethanol. The detector was shielded by a second polarizer, oriented orthogonally to the excitation light, thus reducing the photocurrent due to excitation light leakage on the detector by approximately 25 dB. The fluorescence emission light, which is randomly polarized, is only attenuated by approximately 3 dB. Fluorescence signals from Rhodamine 6G (peak emission wavelength of 570 nm) and fluorescein (peak emission wavelength of 494 nm) dyes were measured in a dilution series in the microfluidic device with emission signals detected by the OPD. A limit-of-detection of 100 nM was demonstrated for Rhodamine 6G, and 10 microM for fluorescein. This suggests that an integrated microfluidic device, with an organic photodiode and LED excitation source and integrated polarizers, can be fabricated to realize a compact and economical lab-on-a-chip for point-of-care fluorescence assays.

Measurement of the pressure dependence of emission induced by electrons

The fluorescence detection of ultra high energy ({approx}> 10{sup 18} eV) cosmic rays requires a detailed knowledge of the fluorescence light emission from nitrogen molecules, which are excited by the cosmic ray shower particles along their path in the atmosphere. We have made a precise measurement of the fluorescence light spectrum excited by MeV electrons in dry air. We measured the relative intensities of 34 fluorescence bands in the wavelength range from 284 to 429 nm with a high resolution spectrograph. The pressure dependence of the fluorescence spectrum was also measured from a few hPa up to atmospheric pressure. Relative intensities and collisional quenching reference pressures for bands due to transitions from a common upper level were found in agreement with theoretical expectations. The presence of argon in air was found to have a negligible effect on the fluorescence yield. We estimated that the systematic uncertainty on the cosmic ray shower energy due to the pressure dependence of the fluorescence spectrum is reduced to a level of 1% by the AIRFLY results presented in this paper.

Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

Evidence for Tetraphenylporphyrin Monoacids

Upon dilution from a concentrated solution in dichloromethane, the diacid form of tetraphenylporphyrin {H(4)TPP(X)(2)} (X = Cl, PF(6) and tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, TFPB) affords eventually the unprotonated free base species H(2)TPP. At a difference of chloride, in the case of PF(6)(-) and TFPB(-) anions the conversion occurs with the intermediacy of a species, which has been assigned to a monoacid derivative on the basis of UV/vis absorption, fluorescence emission (static and dynamic), and resonance light scattering. Ground-state gas-phase geometries have been calculated both for the diacid {H(4)TPP(PF(6))(2)} and monoacid {H(4)TPP(PF(6))(2)} and {H(3)TPP(Cl)} species at the DFT/BP86 level of theory. TDDFT calculations using different functionals (BP86, SAOP, and B3LYP) have been exploited to provide electronic vertical excitation energies and oscillator strengths, yielding a remarkably good description of the optical spectra for these compounds and supporting the identification of the monoacid species. Gas-phase thermodynamic calculations on the chloride species provide an estimate of the Gibbs free energy changes associated with the two protonation steps, supporting the observed different behavior of this anion with respect to PF(6)(-) and TFPB(-).

Measurement of the pressure dependence of air fluorescence emission induced by electrons

The fluorescence detection of ultra high energy (≳10 18 eV) cosmic rays requires a detailed knowledge of the fluorescence light emission from nitrogen molecules, which are excited by the cosmic ray shower particles along their path in the atmosphere. We have made a precise measurement of the fluorescence light spectrum excited by MeV electrons in dry air. We measured the relative intensities of 34 fluorescence bands in the wavelength range from 284 to 429 nm with a high resolution spectrograph. The pressure dependence of the fluorescence spectrum was also measured from a few hPa up to atmospheric pressure. Relative intensities and collisional quenching reference pressures for bands due to transitions from a common upper level were found in agreement with theoretical expectations. The presence of argon in air was found to have a negligible effect on the fluorescence yield. We estimated that the systematic uncertainty on the cosmic ray shower energy due to the pressure dependence of the fluorescence spectrum is reduced to a level of 1% by the AIRFLY results presented in this paper.

Model of Fluorescence Intermittency in Single Enzymes

The intermittent emission of fluorescent light from single enzymes, quantum dots, and other nanoscale systems is often characterized by statistical correlations in the emitted signal. A one-dimensional model of such correlations in enzymes, based on a model of protein conformational fluctuations developed by Kou and Xie (Phys. Rev. Lett. 2004, 93, 180603), is formulated in the present paper in terms of the dynamics of a particle moving stochastically between "on" and "off" states under the action of fractional Gaussian noise. The model yields predictions for the short and long time behavior of the following quantities: the time correlation function, C(t), of the fluctuations of the signal intensity, the distribution, f(t), of time intervals between intensity fluctuations, and the Mandel parameter, Q(t), describing the extent of bunching or anti-bunching in the signal. At short times, C(t) and f(t) are found to decay exponentially, while, at long times, they are found to decay as power laws, the exponents being functions solely of the nature of the temporal correlations in the noise. The results are in good qualitative agreement with results from single-molecule experiments on fluorescence intermittency in the enzyme cholesterol oxidase carried out by Xie and co-workers (Science 1998, 282, 1877). The Mandel parameter, Q(t), for this model is positive at short and long times, indicating super-Poisson statistics in these limits, consistent with bunching of the fluorescent signal.

Development of a Method Using Image Analysis for the Measurement of Cellulose‐Binding Domains Adsorbed onto Cellulose Fibers

The surface concentration of CBD-FITC conjugates, adsorbed on cellulose fibers, was determined by image analysis. The program consists of two scripts, the first dedicated to the elaboration of the calibration curve. The emission of fluorescent light, detected by image analysis, is correlated with the concentration of CBD solutions. This calibration is then used (second script) to determine the concentration of CBDs adsorbed on cellulosic fibers. This method allows the direct estimation of the surface concentration of adsorbed CBDs, which usually is not accurately calculated from depletion studies, since the surface area is hardly known. By observing different spots in the surface of the fibers, site-specific information is obtained. It was verified that the physically heterogeneous fibers exhibit different amounts of adsorbed CBDs.

Influence of the chemical structure of dithiocarbamates with different R groups on the reversible addition‐fragmentation chain transfer polymerization

AbstractFour dithiocarbamates, carbazole‐9‐carbodithioic acid benzyl ester (R1), carbazole‐9‐carbodithioic acid naphthalen‐1‐ylmethyl ester (R2), 2‐(carbazole‐9‐carbothioylsulfanyl)‐2‐methyl‐propionic acid ethyl ester (R3), and (carbazole‐9‐carbothioylsulfanyl)‐phenyl‐acetic acid methyl ester (R4), were synthesized and used to the reversible addition‐fragmentation chain transfer (RAFT) polymerizations of styrene (St), methyl methacrylate (MMA), and methyl acrylate (MA), respectively. The influence of chemical structure of dithiocarbamates with different R groups on the RAFT polymerizations was investigated. The results showed that the four RAFT agents were effective RAFT agents for the polymerizations of styrene or MA, and that the polymerizations were well‐controlled with the characteristics of controlled/“living” polymerization. The polymerization rate of styrene with thermal initiation was markedly influenced by the chemical structures of the group R in dithiocarbamates, and decreased in the order of R3 > R2 > R4 > R1. For the polymerization of MA, the efficiency of RAFT agents was in the following order: R2–R3 > R1 > R4. However, they were not efficient enough to control the polymerization of MMA. The obtained polystyrene (PSt) with carbazole group labeled strongly absorbed UV light at 294 nm and emitted fluorescent light in N,N‐dimethyl formamide (DMF). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 982–988, 2007

Imaging Farnesyl Protein Transferase Using a Topologically Activated Probe

We report on the development and application of a "smart" activatable peptide-based probe for detection of Ras-related farnesyl protein transferase (FPT). Upon farnesylation by FPT, the probe was brought close to a hydrophobic milieu and as a consequence emitted fluorescent light that could be detected by several media, such as fluorescence microscopy, a plate reader, and an optical imaging system. A FPT activity assay confirmed the specificity of the probe (IC50 = 1.2 muM) for FPT compared to that of the native peptide (IC50 = 0.17 muM). In addition, the probe has remarkable binding constant, Kd = 26 nM. The specificity of enzyme activation was proved in pure enzyme assays as well as in cell-based assays. Furthermore, the fluorescent enhancement of the probe was 30-fold stronger than the control peptide in a live cell assay.

Auto fluorescence of intervertebral disc tissue: a new diagnostic tool

The paper reports on auto fluorescence phenomena of inter-vertebral human discs. It systematically investigates the auto fluorescence effects of ex vivo disc specimen and reports on surgical cases to demonstrate the potential value of the new method. The paper offers biologic explanations of the phenomenon and discusses the potential value of the UV auto fluorescence technique as a diagnostic tool. Intra- and postoperative observations are made by a surgical microscope with an integrated UV light source. Quantitative measurements were carried out using a photon counter and a spectrometer ex vivo. The auto fluorescence phenomenon allows the differentiation of traumatized and degenerated disc tissue intraoperatively in some cases, it allows the differentiation of bony and collagen endplate in cervical disc surgery. The source of the auto fluorescent light emission are amino acids of the collagen molecules. The proteoglycan components and the liquid components of the disc do not show relevant auto fluorescence. Emission wavelength of disc material is equivalent to color perception. It differs due to different collagen composition of the intervertebral disc components from yellow-green to blue-green and can be visualized in situ by naked eye.UV-auto fluorescence of inter-vertebral discs is a new clinical tool that has the potential to differentiate disc material from the anatomical surrounding, to distinguish between different fractions of the disc and to give information on the quality and status of the disc material. Since the technology has just emerged, it needs further investigations to quantify the clinical observations reported in this paper.

Fabricaton of Poly(dimethylsiloxane) Microlens for Laser-Induced Fluorescence Detection

In laser-induced fluorescence (LIF) detection, the focusing of the excitation laser beam into a focal point of the channel in a microfluidic chip is important to obtain the largest excitation intensity, and consequently to obtain the emitted fluorescence light collected to the photodetector with a high efficiency. In this paper, we present a poly(dimethylsiloxane) (PDMS) microfluidic chip consisting of an integrated PDMS microlens. The PDMS microlens was fabricated by the photoresist reflow and replica molding method. This fabrication technique is simple and has a good property in terms of the microlens and a high-dimensional accuracy. We showed that fluorescence intensities using the microlens were found to be approximately four times higher than those without the microlens. The microlens integrated on the microfluidic channel was verified to improve fluorescence intensity, and consequently signal to noise ratio.

Laser-Induced Fluorescence Detection System for Microfluidic Chips Based on an Orthogonal Optical Arrangement

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.

Impact of varying atmospheric profiles on extensive air shower observation: Fluorescence light emission and energy reconstruction

Several experiments measure the fluorescence light produced by extensive air showers in the atmosphere. This light is converted into a longitudinal shower profile from which information on the primary energy and composition is derived. The fluorescence yield, as the conversion factor between light profile measured by EAS experiments and physical interpretation of showers, has been measured in several laboratory experiments. The results, however, differ considerably. In this article, a model calculation of the fluorescence emission from relevant band systems of nitrogen in dependence on wavelength and atmospheric conditions is presented. Different calculations are compared to each other in combination with varying input parameters. The predictions are compared with measurements and the altitude dependence of the fluorescence yield is discussed in detail.

Shower size parameter as an estimator of extensive air shower energy in fluorescence telescopes

The fluorescence technique has been successfully used to detect ultrahigh energy cosmic rays by indirect measurements. The underlying idea is that the number of charged particles in the atmospheric shower, i.e, its longitudinal profile, can be extracted from the amount of emitted nitrogen fluorescence light. However the influence of shower fluctuations and the very possible presence of different nuclear species in the primary cosmic ray spectrum makes the estimate of the shower energy from the fluorescence data analysis a difficult task. We investigate the potential of shower size at maximum depth as estimator of shower energy. The detection of the fluorescence light is simulated in detail and the reconstruction biases are carefully analyzed. We extend our calculations to both HiRes and EUSO experiments. This approach has shown some advantages to the reconstruction of the energy when compared to the standard analysis procedure.

Role of Counteranions in Acid-Induced Aggregation of Isomeric Tetrapyridylporphyrins in Organic Solvents

The effect of adding various kinds of acids HX (X = Cl, Br, I, CF(3)COO, CF(3)SO(3), TFPB ((3,5-(CF(3))(2)C(6)H(3))(4)B) to isomeric tetra(2-pyridyl)porphyrin, tetra(3-pyridyl)porphyrin, and tetra(4-pyridyl)porphyrin (TpyP(2), TpyP(3), and TpyP(4)) in dichloromethane solution has been investigated through the combined use of UV/vis absorption, fluorescence emission, and resonance light scattering (RLS) techniques. The experimental evidence points to a marked dependence of the protonation and aggregation behavior on the nature of both acids and porphyrins. In general, three different trends can be recognized: (i) formation of a fully protonated species, followed by aggregation; (ii) formation of a tetraprotonated species, which aggregates and, on further addition of acid, disaggregates; and (iii) protonation of the four pyridyl moieties, leading to a tetraprotonated ion pair, in the unique case of the bulky TFPB(-) anion. In all cases, the protonated species and the resulting aggregates exhibit spectroscopic features that are markedly influenced by the nature of the counteranions. A model for J-aggregation has been proposed on the basis of an interplay of hydrogen bonding, electrostatic interactions, and dispersive interactions. Kinetic control of the aggregation process allows for a fine-tuning of the spectroscopic properties of the final aggregated species.

Tomographic fluorescence imaging in tissue phantoms: a novel reconstruction algorithm and imaging geometry

A novel image reconstruction algorithm has been developed and demonstrated for fluorescence-enhanced frequency-domain photon migration (FDPM) tomography from measurements of area illumination with modulated excitation light and area collection of emitted fluorescence light using a gain modulated image-intensified charge-coupled device (ICCD) camera. The image reconstruction problem was formulated as a nonlinear least-squares-type simple bounds constrained optimization problem based upon the penalty/modified barrier function (PMBF) method and the coupled diffusion equations. The simple bounds constraints are included in the objective function of the PMBF method and the gradient-based truncated Newton method with trust region is used to minimize the function for the large-scale problem (39919 unknowns, 2973 measurements). Three-dimensional (3-D) images of fluorescence absorption coefficients were reconstructed using the algorithm from experimental reflectance measurements under conditions of perfect and imperfect distribution of fluorophore within a single target. To our knowledge, this is the first time that targets have been reconstructed in three-dimensions from reflectance measurements with a clinically relevant phantom.

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 μg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells

A Laser Scanning Cytometry-based method was developed for identification of transgenic mice expressing green fluorescent protein (GFP) using minute amounts of peripheral blood. The difference between the autofluorescence of cells not expressing GFP and the fluorescence of GFP expressing cells after excitation with Ar-ion laser (wavelength 488 nm) and detection of emitted fluorescent light in the green channel was high enough for unambiguous identification of the GFP expressing mice. The sensitivity of this method was estimated 1:10 4 for detection of rare GFP expressing cells under the conditions used. This sensitivity should be sufficient for many studies on microchimerism. Because of the possibility for relocation of the cells, this method will be particularly useful for characterizing the cells with high GFP expression using other markers of cell phenotype or conventional morphological analysis.