Front Face Excitation Research Articles

Characterization of activated sludge in wastewater treatment processes using front-face excitation–emission matrix (FF-EEM) fluorescence spectroscopy

The novel front-face fluorescence excitation–emission matrix (FF-EEM) spectroscopy was applied to in situ and online analyze the activated sludge from wastewater treatment processes to probe its biomass and bioactivity.

Long wavelength fluorescence lifetime standards for front-face fluorometry.

With the increased development and use of fluorescence lifetime-based sensors, fiber optic sensors, fluorescence lifetime imaging microscopy (FLIM), and plate and array readers, , calibration standards are essential to ensure the proper function of these devices and accurate results. For many devices that utilize a "front face excitation" geometry where the excitation is nearly coaxial with the direction of emission, scattering-based lifetime standards are problematic and fluorescent lifetime standards are necessary. As more long wavelength (red and near-infrared) fluorophores are used to avoid background autofluorescence, the lack of lifetime standards in this wavelength range has only become more apparent . We describe an approach to developing lifetime standards in any wavelength range, based on Förster resonance energy transfer (FRET). These standards are bright, highly reproducible, have a broad decrease in observed lifetime, and an emission wavelength in the red to near infrared making them well suited for the laboratory and field applications as well. This basic approach can be extended to produce lifetime standards for other wavelength regimes.

Thermophysical properties mapping in semi-infinite longitudinally cracked plates by temperature image processing

Thermophysical properties mapping in a solid plate from transient temperature imaging analysis is widely developed, but most usual methods are based on very restrictive assumptions such as the independence of the pixels (local one-dimensional diffusion in the sample). The challenge envisioned in this study is to take into account the three-dimensional spatial correlation between pixels. Moreover, the estimation of the thermophysical properties field from two-dimensional measurements is an ill-posed problem, and the practical implementation of the estimation is affected by the large amount of data to be processed. A semi-numerical analysis with spatially random front face excitation is proposed here in order to overcome such problems. A theoretical new method is proposed for the detection of cracks laid perpendicular to the excitation surface. The estimation method is implemented from a spatial autoregressive method.

Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces

We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.

Fluorescence Lifetime Distributions of Labeled Amorphous Polymers in Bulk

Polystyrene, poly(methylmethacrylate), poly(cyclohexyl methacrylate) and poly(1-butylmethacrylate) were labeled with anthryl groups by copolymerization. Films of labeled polymers as well as blends of them with unlabeled polystyrene samples of different polydispersity and molecular weight, were prepared by solvent casting. The time resolved emission of anthryl groups in those films was measured by Single Photon Counting with front face excitation at the standard 30° incident angle and with much lower incident angles to photoselect chromophores on the polymer-air surface. Fluorescence decays were fitted with bimodal fluorescence lifetime distributions, which were analyzed taking into consideration some polymer characteristics and the compatibility of polymer blends. It was thus concluded that solid-like and liquid-like environments are both in the bulk and in the surface of the film although liquid-like domains are more frequent on the surface than in bulk. Polymer Tg determines the position of the two modes and polydispersity justify the broadness of the modes in the fluorescence lifetime distribution.

Photodegradation of Pyrene-Labeled Polystyrene Adsorbed on Silica Surface in Chloroform

Photodegradation of Pyrene-Labeled Polystyrene Adsorbed on Silica Surface in Chloroform

Mobility of knots in polyethylacrylate networks

The steady state fluorescence anisotropy of the chromophore dialkylbenzene, which constitutes the crosslinks of polyethylacrylate and polymethylmethacrylate networks, has been investigated over a range of temperatures, from 20 to 80°C. In polymethylmethacrylate networks the limit anisotropy of dialkylbenzene (r o =0.198) and vinylphenyl side groups of the elastic chains (r o =0.304) have been determined by applying a correction method (previously reported) to discount artifacts typical of front face excitation. In polyethylacrylate networks it was found that the mobility of knots does not change appreciably with temperature even 100°C above the glass transition of the system (about 10°C). It was concluded that polymer dynamics that affect the mobility of knots is different of that determining the motions of the elastic chains.

Influence of adventitious light scattering on fluorescence anisotropy measurements with front-face excitation. Simulation and correction

Abstract Fluorescence anisotropy spectra of a generic chromophore have been simulated for different proportions of adventitious light scattering with respect to fluorescence. The spectral overlap of Rayleigh light scattering and stray light with fluorescence causes an increase in the apparent anisotropy with respect to the real fluorescence anisotropy and curvature of the anisotropy spectra. In the presence of up to about 10% fluorescence contamination, the largest effect of these artefacts occurs for true anisotropies close to 0.25 and the least for anisotropies close to −0.2. The apparent anisotropy, determined at the fluorescence maximum or at the anisotropy minimum, increases almost linearly with f (the fraction of stray light with respect to the maximum fluorescence intensity) for low f values ( f o - and m -xylene in a polymeric matrix with front-face excitation.

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.

Practical and Convenient 355-nm and 337-nm Sharp-Cut Filters for Multichannel Raman Spectroscopy

The interference from Rayleigh and other elastic scattering is a serious problem for multichannel Raman spectroscopy. This problem becomes more serious if the surface subjected to the excitation reflects stray light, particularly in front-face excitation and in systems using rotating samples or applying the flow technique to avoid local heating and accumulation of the photoproduct. In order to eliminate this problem the conventional multichannel Raman spectrometer uses a specially designed triple polychromator equipped with a subtractive double-grating filter stage. However, due to the complicated optical arrangements, including three gratings and at least six mirrors, the throughput of the instrument is usually low. This is a severe disadvantage if one needs to detect extremely weak Raman signals. This is particularly true when time-resolved resonance Raman (TRR) spectroscopy is performed. In order to have an optimum TRR signal, the ideal method is to apply a single spectrograph equipped with a sharp line elimination filter tuned to the wavelength of the exciting line. Several filters such as chevron filters and colloid filters have been designed for this purpose. Unfortunately, most of these filters are only applicable in the visible region. In addition, the requirement of a high degree of collimation also reduces the throughput of the signal. Several commercially available sharp-cut long-wavelength bandpass filters and a recently developed J-aggregate filter applying organic dye molecules are also useful for the elimination of exciting lines in the visible region.

PHOTOFRIN II UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS. FLUORESCENCE AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS ON POST‐MORTEM AORTA

Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematoporphyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.