Spectroscopic study of a novel bis(heptamethine cyanine) dye and its interaction with human serum albumin

A newly synthesized near-infrared (NIR) bis(heptamethine cyanine) dye 7 was evaluated for its utility as a non-covalent label for proteins. This dye forms inter- and intramolecular H-aggregates in polar solvents, even at very low concentrations. The intramolecular dimeric form of the dye can be described as a clam-shell complex with two interacting hydrophobic carbocyanine moieties. In this intramolecular H-aggregate, the chromophore has a low extinction coefficient and low fluorescence quantum yield. In aqueous solution, in the absence of human serum albumin (HSA), dye 7 has characteristic absorption bands at 792 and 435 nm, and its fluorescent emission is significantly diminished in comparison to that in methanol or when compared to its monomeric equivalent 5. Dye 7 seems to be more advantageous than its monomeric counterpart 5 as a non-covalent label for biomolecules. Upon addition of HSA, the H and D bands are decreased and the monomeric band is increased, with concomitant increase in fluorescence intensity, suggesting that clam-shell H-aggregates open up in the complex with HSA. The binding stoichiometry is 1:1. The main advantage of this dimeric dye as a non-covalent label is that the free dye has negligible fluorescence.     

Spatial distribution of two-photon-excited fluorescence in scattering media

Spatial distribution of two-photon-excited fluorescence (TPF) for dye beneath the surface of a highly scattering medium was investigated with picosecond laser pulses at 1064 nm. The active scattering media consisted of a suspension of polystyrene particles in a solution of Rhodamine 590 Tetrafluoroborate dye. With the increase of scattering strength of the medium, the location of the maximum TPF intensity was found to move closer to the surface away from the focal region; the intensity of TPF was not confined to the focal region as in the case of nonscattering medium but was more evenly distributed. The spatial resolution of nonlinear optical microscopy for probing a scattering medium is degraded. Taking into account the scattering of the medium qualitatively explains the observed TPF spatial distribution.     

Comparison of surface-enhanced resonance Raman scattering and fluorescence for detection of a labeled antibody

A comparison is made of the quantitative detection of a labeled antibody by surface-enhanced resonance Raman scattering (SERRS) and by fluorescence using the same instrument with the same laser excitation source. The area under the curve for the fluorescence band is greater than for any single peak in the SERRS spectrum, but the broad fluorescence band is more difficult to discriminate from the background at low concentrations. Using the peak height of one SERRS band and the peak height at the fluorescence maximum, the detection limit for SERRS was lower (1.19 x 10-11 mol.dm-3) than that obtained using fluorescence (3.46 x 10-10 mol.dm-3). The SERRS detection limit is calculated for the concentration of the sample added, but compared to fluorescence, there is an additional dilution step due to the addition of the colloid and the extent of this dilution is dependent on assay format. For comparison with the detection limits found earlier with labeled oligonucleotides, SERRS was remeasured with a 10 s accumulation time, and the final concentration in the cuvette after colloid addition and before any adsorption to the silver was used to calculate a detection limit of 2.79 x 10-13 mol.dm-3. This is comparable to the detection limit found using a similar SERRS procedure for an oligonucleotide labeled with the same dye. This experiment is dependent on many parameters that could affect this result, including the nature of the SERRS substrate, the excitation wavelength, and the dye chosen. However, the result indicates that SERRS can give assay sensitivities comparable or better than fluorescence for quantitative direct assay determination, suggesting that the much greater potential for multiple analyte detection could be exploited.     

High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy

Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below approximately 10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 ns, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.     

Side illumination fluorescence emission characteristics from a dye doped polymer optical fiber under two-photon excitation

Two-photon excited (TPE) side illumination fluorescence studies in a Rh6G-RhB dye mixture doped polymer optical fiber (POF) and the effect of energy transfer on the attenuation coefficient is reported. The dye doped POF is pumped sideways using 800 nm, 70 fs laser pulses from a Ti:sapphire laser, and the TPE fluorescence emission is collected from the end of the fiber for different propagation distances. The fluorescence intensity of RhB doped POF is enhanced in the presence of Rh6G as a result of energy transfer from Rh6G to RhB. Because of the reabsorption and reemission process in dye molecules, an effective energy transfer is observed from the shorter wavelength part of the fluorescence spectrum to the longer wavelength part as the propagation distance is increased in dye doped POF. An energy transfer coefficient is found to be higher at shorter propagation distances compared to longer distances. A TPE fluorescence signal is used to characterize the optical attenuation coefficient in dye doped POF. The attenuation coefficient decreases at longer propagation distances due to the reabsorption and reemission process taking place within the dye doped fiber as the propagation distance is increased.     

Background correction by wavelength modulation for pulsed-laser-excited atomic fluorescence spectrometry.

Instrumentation was constructed to modulate the dye laser wavelength for background correction in laser-excited atomic fluorescence spectrometry (LEAFS). To achieve wavelength modulation a piezoelectric pusher was used to drive the wavelength tuning mirror in a laboratory-constructed grazing incidence dye laser. The laser pulses were synchronized with the piezoelectric pusher movement so that alternate laser pulses measured the atomic fluorescence signal at the analytical atomic spectral line (on-line) and the background signal at a wavelength displaced to one side of the atomic line (off-line). The background-corrected signal was obtained by subtracting the off-line "background" from the on-line "signal plus background". The spectral line width (fwhm) of the dye laser was 0.003 nm, while the wavelength modulation interval was controllable over the range from 0 to 0.2 nm with a spectral resolution limited only by the spectral line width of the laser. This type of background correction could, in principle, be applied to other types of tunable lasers such as pulsed Ti: sapphire lasers. The performance of background correction by wavelength modulation (WM) was demonstrated by measurement of sodium resonance fluorescence in an air-acetylene flame and by thallium nonresonance fluorescence in a graphite furnace. The experimental data indicated that the wavelength modulation corrected, effectively and quantitatively, for flame background, blackbody emission from a graphite furnace, and scatter of laser radiation off aluminum chloride (1 mg/mL as AI) matrix particles in both the furnace and the flame. Analytical results were in good agreement with certified values for the determination of sodium in standard reference materials by the use of modulated LEAFS.     

Studies on the two-photon pumped upconverted fluorescence and superradiance of a new organic dye material in solutions

The linear and nonlinear optical properties of a new organic dye, trans-4-[p-(N-ethyl-N-ethylamino)-styryl]-N-methyl-pyridinium tris(thiocyanato) cadmates (II), are reported in this paper. When pumped with a picosecond laser at the wavelength range of 850-1200 nm, intense upconversion fluorescence can be obtained. The upconversion efficiencies at different pump energies were measured when pumped with a 1064-nm laser beam from a mode-locked Nd:YAG laser. The highest upconversion efficiencies were measured to be 5.8% and 7.6% in dimethyl formamide (DMF) and methanol. The lifetime of the dye in DMF was measured to be 75 ps. The strongest nonlinear absorption was at the wavelength of 940 nm, and the highest upconversion efficiency was at the wavelength of 1030 nm. The difference of the two wavelengths was caused by excited state absorption in the dye at wavelengths shorter than 1000 nm. The dye solution in DMF and methanol show a clear optical power limiting effect.     

Multiple kinks in lamellar linear polyethylene

Injection-moulded specimens made of linear polyethylene with preferred orientation of the molecular chains (c-axes) in the direction of injection were deformed in a bending test. Ultra-thin sections of the deformed material were made following chlorosulfonization. Examination using transmission electron microscopy revealed that deformational and superstructural elements can be elucidated simultaneously using this method. Deformation in the observed regions with multiple kink bands is found to be inhomogeneous and concentrated in two shearing processes: one along the borders of the kink band and the other within a kink band at an acute angle to the border of the band. It must be concluded from the formation of the lamellae in the kink bands that deformation, even within a kink band, is inhomogeneous and is predominantly concentrated in the shear zones, while the lamellar stacks lying between the shear zones appear under electron microscopy, to be nearly undeformed.     

Two-photon nitric oxide laser-induced fluorescence measurements in a diesel engine

A two-photon nitric oxide (NO) laser-induced fluorescence (LIF) technique was developed and applied to study in-cylinder diesel combustion. The technique prevents many problems associated with in-cylinder, single-photon NO planar-laser-induced fluorescence measurements, including fluorescence interference from the Schumann-Runge bands of hot O2, absorption of a UV excitation beam by in-cylinder gases, and difficulty in rejecting scattered laser light while simultaneously attempting to maximize fluorescence signal collection. Verification that the signal resulted from NO was provided by tuning of the laser to a vibrational off-resonance wavelength that showed near-zero signal levels, which resulted from either fluorescence or interference at in-cylinder pressures of as much as 20 bar. The two-photon NO LIF signal showed good qualitative agreement with NO exhaust-gas measurements obtained over a wide range of engine loads.     

Two-photon fluorescence imaging super-enhanced by multishell nanophotonic particles, with application to subcellular pH

A novel nanophotonic method for enhancing the two-photon fluorescence signal of a fluorophore is presented. It utilizes the second harmonic (SH) of the exciting light generated by noble metal nanospheres in whose near-field the dye molecules are placed, to further enhance the dye's fluorescence signal in addition to the usual metal-enhanced fluorescence phenomenon. This method enables demonstration, for the first time, of two-photon fluorescence enhancement inside a biological system, namely live cells. A multishell hydrogel nanoparticle containing a silver core, a protective citrate capping, which serves also as an excitation quenching inhibitor spacer, a pH indicator dye shell, and a polyacrylamide cladding are employed. Utilizing this technique, an enhancement of up to 20 times in the two-photon fluorescence of the indicator dye is observed. Although a significant portion of the enhanced fluorescence signal is due to one-photon processes accompanying the SH generation of the exciting light, this method preserves all the advantages of infrared-excited, two-photon microscopy: enhanced penetration depth, localized excitation, low photobleaching, low autofluorescence, and low cellular damage.     

Measurement of absolute distance employing a tunable CW dye laser

With a tunable CW dye laser oscillating in a single longitudinal mode, measurement of an absolute distance is demonstrated with the method of excess fractions. Five beams which have different wavelengths are emitted sequentially from the dye laser, and the interferometric phase is measured for each wavelength. An interferometric order number for a wavelength can be calculated from values of wavelengths and phases. Then a precise value of length is obtained. This method is similar to measuring distances by using group delay as used in VLBI and microwave ranging. The measured accuracy was within ±8.8 nm between 0 and 10 mm (at an absolute distance of 0.1-10.1 mm)     

Ultra violet resonance Raman spectroscopy in lignin analysis: determination of characteristic vibrations of p-hydroxyphenyl, guaiacyl, and syringyl lignin structures

Raman spectroscopy of wood and lignin samples is preferably carried out in the near-infrared region because lignin produces an intense laser-induced fluorescence background at visible excitation wavelengths. However, excitation of aromatic and conjugated lignin structures with deep ultra violet (UV) light gives resonance-enhanced Raman signals while the overlapping fluorescence is eliminated. In this study, ultra violet resonance Raman (UVRR) spectroscopy was used to define characteristic vibration bands of model compounds of p-hydroxyphenyl, guaiacyl, and syringyl lignin structures at three excitation wavelengths (229, 244, and 257 nm). The intensities of each band, relative to the intensity of the aromatic vibration band at 1600 cm-1, were defined and the most suitable excitation wavelength was suggested for each structure. p-Hydroxyphenyl structures showed intensive characteristic bands at 1217-1214 and 1179-1167 cm-1 with excitation at 244 nm, whereas the bands of guaiacyl structures were more intensive with 257 nm excitation. Most intensive characteristic bands of guaiacyl structures were found at 1289-1279, 1187-1185, 1158-1155, and 791-704 cm-1. Syringyl structures had almost identical spectra with 244 and 257 nm excitations with characteristic bands at 1514-1506, 1333-1330, and 981-962 cm-1. The characteristic bands of the three structural units were also found from the compression wood, softwood, and hardwood samples, indicating that UVRR spectroscopy can be applied for the determination of chemical structures of lignin.     

Microchip electrophoresis of tagged probes incorporated with one-colored ddNTP for analyzing single-nucleotide polymorphisms

We demonstrate a simple and rapid method for SNP typing, allele frequency determination, and trace mutant analysis that works with even an inexpensive detection system. This method is based on microchip electrophoresis of tagged probes incorporated with one-colored ddNTP (METPOC). The assay uses dye terminator incorporation into a pair of probes of different lengths specific to wild- and mutant-type targets, respectively. They are hybridized to the targets prior to ddNTP-Cy-5 incorporation, which occurs only for a matched probe-target duplex. Because the extension reactions for the two probes are carried out simultaneously in one tube and the products from both probes are analyzed in one channel by one-color fluorescence detection, an accurate comparative analysis of SNPs is possible. SNP typing as well as allele frequency determination in the range above 0.1% can easily be carried out using a commercial microchip electrophoresis system in a few minutes.     

Next-Generation NASA Airborne Oceanographic Lidar System

The complete design and flight test of the next-generation Airborne Oceanographic Lidar (AOL-3) is detailed. The application of new technology has allowed major reductions in weight, volume, and power requirements compared with the earlier AOL sensor. Subsystem designs for the new AOL sensor include new technology in fiber optics, spectrometer detector optical train, miniature photomultiplier modules, dual-laser wavelength excitation from a single small laser source, and new receiver optical configuration. The new design reduced telescope size and maintained the same principal fluorescence and water Raman bands but essentially retained a comparable measurement accuracy. A major advancement is the implementation of single-laser simultaneous excitation of two physically separate oceanic target areas: one stimulated by 532 nm and the other by 355 nm. Backscattered fluorescence and Raman signals from both targets are acquired simultaneously by use of the same telescope and spectrometer-detector system. Two digital oscilloscopes provide temporal- and depth-resolved data from each of seven spectral emission bands.     

Multispectral decomposition for the removal of out-of-band effects of visible/infrared imaging radiometer suite visible and near-infrared bands

The visible/infrared imaging radiometer suite (VIIRS) is now onboard the first satellite platform managed by the Joint Polar Satellite System of the National Oceanic and Atmospheric Administration and NASA. It collects scientific data from an altitude of approximately 830 km in 22 narrow bands located in the 0.4-12.5 μm range. The seven visible and near-infrared (VisNIR) bands in the wavelength interval between 0.4-0.9 μm are known to suffer from the out-of-band (OOB) responses--a small amount of radiances far away from the center of a given band that can pass through the filter and reach detectors in the focal plane. A proper treatment of the OOB effects is necessary in order to obtain calibrated at-sensor radiance data [referred to as the Sensor Data Records (SDRs)] from measurements with these bands and subsequently to derive higher-level data products [referred to as the Environmental Data Records (EDRs)]. We have recently developed a new technique, called multispectral decomposition transform (MDT), which can be used to correct/remove the OOB effects of VIIRS VisNIR bands and to recover the true narrow band radiances from the measured radiances containing OOB effects. An MDT matrix is derived from the laboratory-measured filter transmittance functions. The recovery of the narrow band signals is performed through a matrix multiplication--the production between the MDT matrix and a multispectral vector. Hyperspectral imaging data measured from high altitude aircraft and satellite platforms, the complete VIIRS filter functions, and the truncated VIIRS filter functions to narrower spectral intervals, are used to simulate the VIIRS data with and without OOB effects. Our experimental results using the proposed MDT method have demonstrated that the average errors after decomposition are reduced by more than one order of magnitude.     

Single-molecule identification by spectrally and time-resolved fluorescence detection

A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.     

Fluorescent carbon dots with two absorption bands: luminescence mechanism and ion detection

Herein, we report the synthesis of carbon dots (CDs) with two characterized absorption bands but without excitation wavelength-dependent fluorescence via a one-step hydrothermal method. The structure of CDs was characterized using X-ray photoelectron spectroscopy, high-resolution transmission electron microscopy, Fourier transform infrared, and UV–Vis spectroscopy. The structure and photoluminescence of CDs vary significantly with different raw materials and preparation methods, and the mechanism of luminescence is not clear yet. Hence, we studied the luminescence mechanism behind two characterized absorption bands of CDs using fluorescence quenching method with ninhydrin and several ions as quenchers. The influence of the surface groups of CDs on its photoluminescence properties was also discussed. Ninhydrin and a variety of other ions exhibited different quenching effects on the fluorescence emissions which obtained at the two absorption bands of CDs. Combining with the structure characterization results, it can be concluded that the emission wavelength is mainly determined by the carbon core, while the excitation wavelength is determined by the surface nitrogen-containing groups. (The excitation at 234 nm might be due to the Schiff base structure, while the excitation at 345 nm was mainly due to the amide structure.) Furthermore, based on the interaction of NO₂^? with the surface nitrogen-containing groups of CDs, a quantitative detection method of NO₂^? using CDs was proposed in our study. CDs exhibited high selectivity for NO₂^? at pH 1.6 with good linearity to NO₂^? concentration in the range of 1–10 μM.     

Rapid forensic analysis and identification of ''lilac'' architectural finishes using Raman spectroscopy

The potential of Raman spectroscopy to discriminate between architectural finishes (household paint) has been investigated using a test set of 51 'lilac' paints and three different excitation wavelengths. The spectra obtained with visible excitation typically displayed a series of intense Raman bands on a featureless fluorescence background but the spectra of all the paints studied had essentially identical bands. With 785 nm excitation, although the same bands that dominated the 514 nm spectra were still observed, other bands with comparable intensity also appeared. The two strongest scattering constituents were identified as a dioxazine dye, Violet 23 and beta-Cu(phthalocyanine). A scatter plot of the intensities of marker bands for these constituents (normalized to the strong rutile bands that were always present) showed that, despite the fact that the sample set spanned a wide range of rutile : dioxazine dye : phthalo- cyanine ratios, many of the samples had very similar ratios and could not be discriminated. However, all the samples (even those with similar relative proportions of the main constituents) could be discriminated on the basis of their minor constituents, either by manually measuring band intensities or through the creation and searching of spectral libraries.     

Multi-frequency encoding for fast color flow or quadroplex imaging

Ultrasonic color flow maps are made by estimating the velocities line by line over the region of interest. For each velocity estimate, multiple repetitions are needed. This sets a limit on the frame rate, which becomes increasingly severe when imaging deeper lying structures or when simultaneously acquiring spectrogram data for triplex imaging. This paper proposes a method for decreasing the data acquisition time by simultaneously sampling multiple lines for color flow maps, using narrow band signals with approximately disjoint spectral support. The signals are separated in the receiver by filters matched to the emitted waveforms, producing a number of data sets with different center frequencies. The autocorrelation estimator is then applied to each of the data sets. The method is presented, various side effects are considered, and the method is tested on data from a recirculating flow phantom. A mean standard deviation across the flow profile of 3.1, 2.5, and 2.1% of the peak velocity was found for bands at 5 MHz, 7 MHz, and 9 MHz, respectively. Alternatively, the method can be used for simultaneously sampling data for a color flow map and for multiple spectrograms using different spectral bands. Using three spectral bands, data for a color flow map and two independent spectrograms can be acquired at the time normally spent on acquiring data for a color flow map only. This yields an expansion of triplex imaging called multifrequency quadroplex imaging, which enables study of the flow over an arterial stenosis by simultaneously acquiring spectrograms on both sides of the stenosis, while maintaining the color flow map. The method was tested in vivo on data from the common carotid artery of a healthy male volunteer, both for fast color flow mapping and for multifrequency quadroplex imaging.     

Three-dimensional autofluorescence spectroscopy of rat skeletal muscle tissue under two-photon excitation

We report on three-dimensional autofluorescence spectroscopy obtained from rat skeletal muscle tissue under two-photon excitation by an ultrashort pulsed-laser beam. It is demonstrated that two types of fluorophores within the skeletal muscle tissue can be simultaneously excited with the laser beam at a wavelength of 800 nm. The two fluorophores exhibited unique fluorescence spectral peaks at wavelengths of 450 and 550 nm. These spectroscopic signals can be used to form a three-dimensional image, giving the information about the biochemical makeup of the skeletal muscle tissue.