Rapid Fluorescence EEM Spectroscopy Using Super-Cycle Hadamard-Transform Multiplexing.

Hadamard-transform (HT) multiplexing has recently been applied to increasingly complex spectroscopic techniques. It had been shown that the data acquisition time for fluorescence excitation emission matrix (EEM) spectroscopy can be reduced by 1 or 2 orders of magnitude using HT multiplexing of the excitation light using a programmable light source. In these previous studies, the data acquisition rate had been limited by the time it took to record an EEM, that is, to complete one cycle of multiplexed excitation spectra. The extraction of chemical information, such as concentration and chemical identity, is then obtained from parallel factor (PARAFAC) analysis of the sequence of EEMs. In this contribution, we increase the data acquisition rate by another order of magnitude, limited ultimately by the time it takes to record a single excitation spectrum. Our algorithm is entirely based on improved data processing, that is, it can be applied to previously recorded HT multiplexed data sets. The algorithm is based on three previously unexplored approaches: (1) we perform a PARAFAC multivariate analysis on the raw (multiplexed) data set, (2) the time-independent PARAFAC loading vectors are obtained prior to obtaining the time-dependent score vectors, and (3) when loading vectors are difficult to obtain from the EEMs, we instead use a rolling-average approach to considerably increase the stability of the fit. Analysis of experimental data shows that the scores of fluorescence EEMs with seven excitation wavelengths and over 1000 emission wavelengths can be obtained in less than 20 ms.

Fluorescence Excitation-Emission-Matrix Imaging.

We present a 4-dimensional (4D) fluorescence imaging system in which each of the 65,536 pixels in the image array contains an excitation-emission-matrix spectrum with 31 excitation wavelengths and 8 emission wavelengths (x, y, λexc, λem). Hadamard-transform multiplexing of the excitation light from a 31-channel programmable light source allows for an increase in the data acquisition rate so that each 65,536-pixel image can be obtained within 8 s. The system is demonstrated and characterized using, first, a 4D image of 10 capillaries filled with four dye solutions and their binary and ternary mixtures, and, second, using a sequence of about 100 images of layered fluorescent dye solutions and their changing fluorescence as a function of temperature. Multivariate analysis using parallel factor analysis produces images of the spatial distribution of the fluorophores together with their relative intensity as a function of time.

HPLC-Detector Based on Hadamard-Transform Fluorescence Excitation-Emission-Matrix Spectroscopy.

We present a new rapid-acquisition HPLC detector based on a Hadamard-transform (HT) excitation-emission-matrix (EEM) fluorescence spectrometer allowing the acquisition of two-dimensional spectra at a rate faster than 6 spectra per second (<150 ms per spectrum). The instrument uses discrete ultraviolet light emitting diode (UV LED) light sources which are multiplexed using patterns derived from a Hadamard-matrix and affords faster spectral acquisition compared to conventional sequentially scanning EEM spectrometers. This new programmable light source was combined with a commercial fluorescence spectrometer and integrated as a detector into an HPLC system. We characterize the HT-EEM spectrometer by rapidly separating and detecting a mixture of five different coumarin dyes, with all five analytes eluting in under 2 min. A parallel factor analysis (PARAFAC) algorithm was able to readily separate and identify all coumarin fluorophores without any prior knowledge of the system, even decomposing two coeluting analytes into two distinct PARAFAC components. The HT-EEM spectrometer provides a novel and versatile detection technique suited for rapid online analysis and quantification of analytes in separation methods such as HPLC.

Fabrication and characterization of laser-heated, multiplexed electrospray emitter.

We present a one-step fabrication method for a new multiplexed electrospray emitter with nine parallel micronozzles. The nozzles were formed by wet chemical etching of the end of a microstructured silica fiber containing nine 10 µm flow channels. By carefully adjusting the water flow through the channels while etching, we controlled the shape of the conical micronozzles and were able to obtain conditions under which the micronozzles, together with the flow channels, formed optical micro-axicon lenses. When 1064 nm light was guided through the flow channels and focused by the micro-axicon lenses into the Taylor cones, we were able to increase the desolvation of a model analyte and thereby increased the spray current produced by the emitter. This work paves the way towards a rapidly modulated mass-spectrometry source having a greatly enhanced throughput.

Simultaneous Double-Pass Absorption and Fluorescence Excitation-Emission Matrix Spectroscopy for Measurements of Reaction Kinetics.

A novel Hadamard-transform excitation-emission matrix (EEM) spectrometer generates two-dimensional (2D) fluorescence matrices at a data acquisition rate of over 6 EEMs per minute and with a spectral resolution of 5.3 nm. Using Fresnel reflections from the sample cell, we could record optical transmission spectra synchronously with the 2D EEMs. The spectrometer was integrated into a custom-designed stopped-flow injection device to collect visible absorption and fluorescence EEM spectra of reacting solutions. Two different kinetic studies on two rapidly evolving chemical reactions with multiple overlapping spectral components were conducted by collecting over 8400 absorption spectra and EEMs. The third-order rate constant for the demetalation of chlorophyll-a to pheophytin-a was experimentally determined to be 450 ± 20 M-2·s-1 as derived from a parallel factor (PARAFAC) analysis where absorption and fluorescence data were combined. A PARAFAC analysis of data collected from the insertion of a copper atom into pheophytin-a resulted in several absorbing components and only a single fluorescing component. A reaction model with an association complex and a sitting-a-top (SAT) complex as intermediates explained the absorption data, resulting in a sequence of second-order reactions with rate constants of 4.0 ± 0.4, 2.7 ± 0.3, and 0.28 ± 0.02 M-1·s-1, respectively. The rate constant of the fluorescence decrease was determined to be 1.7 ± 0.2 M-1·s-1, which is consistent with the fluorescence component being attributed to both the pheophytin-a and the association complex.

Two distinct mechanisms upon absorption of volatile organic compounds into siloxane polymers.

The response of polysiloxane materials to volatile organic compounds (VOCs) including benzene, toluene, ethylbenzene, and toluene (BTEX), as well as cyclohexane, acetone, methanol and isopropanol is studied using thin film large-angle refractometry. Refractive index and thickness changes are measured to quantify the diffusion rate and partition coefficients associated with the absorption and desorption of VOC vapours into polydimethylsiloxane (PDMS) and polydiphenylsiloxane (PDPS) - PDMS copolymer films. Absorption of volatile solvent vapours into siloxane polymers is found to follow two distinct mechanisms with different absorption rates. These mechanisms are also associated with different excess volumes of mixing and may be accompanied by a polymer restructuring step.

Fabrication of axicon microlenses on capillaries and microstructured fibers by wet etching.

A facile method is presented for the fabrication of microlenses at the facet of fused silica capillaries and microstructured fibers. After submersion in hydrogen fluoride solution water is pumped slowly through the center hole of the capillary microchannel to create an etchant gradient extending from the capillary axis. The desired axicon angle is generated by adjusting the etching time and/or concentration of the etchant. Similarly, flow- assisted HF etching of a custom microstructured fiber containing nine microchannels produces nine individual microlenses simultaneously at the fiber facet, where each microaxicon lens shows a similar focusing pattern. A theoretical model of the flow-assisted etching process is used to determine the axicon angle and post angle. Also, a simple ray-based model was applied to characterize the focusing properties of the microaxicons in good agreement with experimental observations.

In-fiber Mach-Zehnder interferometer for gas refractive index measurements based on a hollow-core photonic crystal fiber.

We describe an in-fiber interferometer based on a gas-filled hollow-core photonic crystal fiber. Expressions for the sensitivity, figure of merit and refractive index resolution are derived, and values are experimentally measured and theoretically validated using mode field calculations. The refractive indices of nine monoatomic and molecular gases are measured with a resolution of δns < 10-6.

Application of coupled mode theory and coherent superposition theory to phase-shift measurements on optical microresonators.

Several mathematical models exist in the literature to describe the properties of optical resonators. Here, coupled mode theory and coherent superposition theory are compared and their consistency is demonstrated as they are applied to phase-shift cavity ring-down measurements in optical (micro-)cavities. In the particular case of a whispering gallery mode in a microsphere cavity these models are applied to transmission measurements and backscattering measurements through the fiber taper that couples light into the microresonator. It is shown that both models produce identical relations when applied to these traveling wave cavities.

Refractive indices of common solvents and solutions at 1550 nm.

Using a large-angle refractometer, the refractive indices of water, methanol, acetonitrile, acetone, ethanol, formaldehyde solution, 2-propanol, hexanes, 1-propanol, 2-butanol, tetrahydrofuran, dichloromethane, 1,4-dioxane, cyclohexane, N,N-dimethylformamide, ethylene glycol, N,N-dimethylacetamide, chloroform, glycerol, trichloroethylene, dimethylsulfoxide, p-xylene, ethylbenzene, toluene, m-xylene, benzene, and o-xylene were measured at 1550 nm and compared to literature values. In addition, the refractive indices of 48 aqueous sucrose solutions, 34 aqueous sodium chloride solutions, 40 DMSO-water, 40 ethylene glycol-water, and 40 glycerol-water mixtures were also measured at different concentrations and fit with third-order polynomial expressions. This study provides near-infrared refractive indices that were not previously available and may help with the calibration of refractive index sensors that operate at or near 1550 nm.

Quantitative diffusion and swelling kinetic measurements using large-angle interferometric refractometry.

The uptake and release of sorbates into films and coatings is typically accompanied by changes of the films' refractive index and thickness. We provide a comprehensive model to calculate the concentration of the sorbate from the average refractive index and the film thickness, and validate the model experimentally. The mass fraction of the analyte partitioned into a film is described quantitatively by the Lorentz-Lorenz equation and the Clausius-Mosotti equation. To validate the model, the uptake kinetics of water and other solvents into SU-8 films (d = 40-45 µm) were explored. Large-angle interferometric refractometry measurements can be used to characterize films that are between 15 µm to 150 µm thick and, Fourier analysis, is used to determine independently the thickness, the average refractive index and the refractive index at the film-substrate interface at one-second time intervals. From these values the mass fraction of water in SU-8 was calculated. The kinetics were best described by two independent uptake processes having different rates. Each process followed one-dimensional Fickian diffusion kinetics with diffusion coefficients for water into SU-8 photoresist film of 5.67 × 10(-9) cm(2) s(-1) and 61.2 × 10(-9) cm(2) s(-1).

Quantification of different water species in acetone using a NIR-triple-wavelength fiber laser.

A fiber laser using a thulium-doped ZBLAN gain medium was used to generate laser radiation simultaneously at 1461, 1505 and 1874 nm, with > 5 mW output power at each of the wavelengths. The laser was used to quantify the near-infrared absorption of liquid water in acetone. Additionally, near-infrared spectra were recorded using a broad band source and were interpreted using parallel factor (PARAFAC) analysis to rationalize the concentration-dependent peak shifts.

Spatial coating inhomogeneity of highly reflective mirrors determined by cavity ringdown measurements.

The inhomogeneity of high-reflectivity mirror coatings is a potential error source in the application of the cavity ringdown technique. Here, the ringdown times for different transverse modes were recorded. Together with the observed spatial distribution of these modes the ringdown times can be used to approximately locate the position of coating defects. A simple model based on a weighted sum of Hermite-Gaussian mode functions is used to explain the experimental results.

Absorption measurements in liquid core waveguides using cavity ring-down spectroscopy.

Short liquid core waveguides (LCWs) were included into a fiber-loop cavity ring-down absorption spectrometer to reduce the detection limit over, both, single pass absorption in a LCW and cavity-enhanced absorption using a conventional fiber-loop cavity. LCWs of 5 and 10 cm length were interfaced with a pressure-flow system and a multimode fiber-loop cavity using concave fiber lenses with matching numerical apertures and diameters. Two red dyes, Allura Red AC and Congo Red, were detected with a 532 nm pulsed laser at a 5 nM limit of detection in a detection volume of less than 1 µL, corresponding to a minimal detectable absorbance of less than 4 × 10(-4) cm(-1) and a minimal detectable change in absorption cross section, σ(min) = V(det) × Îµ × C(LOD), of about 14 µm(2) (Allura Red AC) and 37 µm(2) (Congo Red).

Monitoring of vapor uptake by refractive index and thickness measurements in thin films.

We report a method for real-time monitoring of vapor uptake by simultaneous detection of the refractive index, n, and thickness, d, of thin transparent films with a precision of δn=10(-4) and δd<100 nm. The setup combines total internal reflection (Abbé) refractometry with an interferometric imaging method. A fast Fourier transform and phase fitting method is applied for accurate and independent determination of refractive indices and thicknesses. While the uptake of acetone vapor by polydimethylsiloxane is investigated, the system is also suited for characterization of other solid and liquid films.

Fabrication and modeling of multimode fiber lenses.

We report on the fabrication, modeling, and experimental verification of the emission of fiber lenses fabricated on multimode fibers in different media. Concave fiber lenses with a radius of 150 µm were fabricated onto a multimode silica fiber (100 µm core) by grinding and polishing against a ruby sphere template. In our theoretical model we assume that the fiber guides light from a Lambertian light source and that the emission cone is governed solely by the range of permitted emission angles. We investigate concave and convex lenses at 532 nm with different radii and in a variety of surrounding media from air (n(0)=1.00) to sapphire (n(0)=1.77). It was found that noticeable focusing or defocusing effects of a silica fiber lens in ethanol (n(0)=1.36) and dimethyl sulfoxide (DMSO) (n(0)=1.48) are only observed when the fiber lens radius was less than the fiber diameter.

Accelerated size-focusing light activated synthesis of atomically precise fluorescent Au22(Lys-Cys-Lys)16 clusters.

Atomically precise metal nanoclusters are promising candidates for various biomedical applications, including their use as photosensitizers in photodynamic therapy (PDT). However, typical synthetic routes of clusters often result in complex mixtures, where isolating and characterizing pure samples becomes challenging. In this work, a new Au22(Lys-Cys-Lys)16 cluster is synthesized using photochemistry, followed by a new type of light activated, accelerated size-focusing. Fluorescence excitation-emission matrix spectroscopy (EEM) and parallel factor (PARAFAC) analysis have been applied to track the formation of fluorescent species, and to assess optical purity of the final product. Furthermore, excited state reactivity of Au22(Lys-Cys-Lys)16 clusters is studied, and formation of type-I reactive oxygen species (ROS) from the excited state of the clusters is observed. The proposed size-focusing procedure in this work can be easily adapted to conventional cluster synthetic methods, such as borohydride reduction, to provide atomically precise clusters.

Fluorescence excitation-emission matrix (EEM) spectroscopy and cavity ring-down (CRD) absorption spectroscopy of oil-contaminated jet fuel using fiber-optic probes.

Excitation emission matrix (EEM) and cavity ring-down (CRD) spectral signatures have been used to detect and quantitatively assess contamination of jet fuels with aero-turbine lubricating oil. The EEM spectrometer has been fiber-coupled to permit in situ measurements of jet turbine oil contamination of jet fuel. Parallel Factor (PARAFAC) analysis as well as Principal Component Analysis and Regression (PCA/PCR) were used to quantify oil contamination in a range from the limit of detection (10 ppm) to 1000 ppm. Fiber-loop cavity ring-down spectroscopy using a pulsed 355 nm laser was used to quantify the oil contamination in the range of 400 ppm to 100,000 ppm. Both methods in combination therefore permit the detection of oil contamination with a linear dynamic range of about 10,000.

Modeling of fiber-optic fluorescence probes for strongly absorbing samples.

The dynamic range of fiber-optic fluorescent probes such as single fibers and fiber bundles is calculated for strongly absorbing samples, such as process liquids, foodstuffs, and lubricants. The model assumes an excitation beam profile based on a Lambertian light source and uses analytical forms of the collection efficiency, followed by an Abel transformation and numerical integration. It is found that the effect of primary absorption of the excitation light and secondary absorption of the fluorescence is profound. For fiber bundles and bifurcated fiber probes, the upper accessible concentration limit is roughly given by the absorption length of the primary and secondary absorption. Fluorescence detectors that are placed at right angles to the excitation beam axis or collinear to the beam axis are equally strongly affected by secondary absorption. A probe in which the same fiber is used for excitation and for collection of the fluorescence emerges as the fiber probe with the largest accessible concentration range.

Simultaneous and continuous multiple wavelength absorption spectroscopy on nanoliter volumes based on frequency-division multiplexing fiber-loop cavity ring-down spectroscopy.

We demonstrate a method for measuring optical loss simultaneously at multiple wavelengths with cavity ring-down spectroscopy (CRD). Phase-shift CRD spectroscopy is used to obtain the absorption of a sample from the phase lag of intensity modulated light that is entering and exiting an optical cavity. We performed dual-wavelength detection by using two different laser light sources and frequency-division multiplexing. Each wavelength is modulated at a separate frequency, and a broadband detector records the total signal. This signal is then demodulated by lock-in amplifiers at the corresponding two frequencies allowing us to obtain the phase-shift and therefore the optical loss at several wavelengths simultaneously without the use of a dispersive element. In applying this method to fiber-loop cavity ring-down spectroscopy, we achieve detection at low micromolar concentrations in a 100 nL liquid volume. Measurements at two wavelengths (405 and 810 nm) were performed simultaneously on two dyes each absorbing at mainly one of the wavelengths. The respective concentrations could be quantified independently in pure samples as well as in mixtures. No crosstalk between the two channels was observed, and a minimal detectable absorbance of 0.02 cm(-1) was achieved at 405 nm.