Steven J. Choquette

Relative Intensity Correction of Raman Spectrometers: NIST SRMs 2241 through 2243 for 785 nm, 532 nm, and 488 nm/514.5 nm Excitation

Standard Reference Materials® SRMs 2241 through 2243 are certified spectroscopic standards intended for the correction of the relative intensity of Raman spectra obtained with instruments employing laser excitation wavelengths of 785 nm, 532 nm, or 488 nm/514.5 nm. These SRMs each consist of an optical glass that emits a broadband luminescence spectrum when illuminated with the Raman excitation laser. The shape of the luminescence spectrum is described by a polynomial expression that relates the relative spectral intensity to the Raman shift with units in wavenumber (cm−1). This polynomial, together with a measurement of the luminescence spectrum of the standard, can be used to determine the spectral intensity-response correction, which is unique to each Raman system. The resulting instrument intensity-response correction may then be used to obtain Raman spectra that are corrected for a number of, but not all, instrument-dependent artifacts. Peak area ratios of the intensity-corrected Raman spectrum of cyclohexane are presented as an example of a methodology to validate the spectral intensity calibration process and to illustrate variations that can occur in this measurement.

Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis

Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 mumol/l ethidium bromide (EB) and then exposed to 100 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.

Embossable Grating Couplers for Planar Waveguide Optical Sensors

Planar optical waveguides are an attractive tool for use in analytical chemistry and spectroscopy. Although similar to fiber optics, planar waveguides have been slow to be commercially accepted due to the difficulty of coupling light into the guide. Generally, prism coupling is the method of choice in the laboratory, as efficiencies approaching 80% can be reached. However, prisms are impractical for routine use for several reasons:  expensive positioning equipment is required, coupled power is sensitive to environmental fluctuations, and prism coupling prohibits the fabrication of a truly planar device. The use of thin gratings on the surface of the waveguide allows for a two-dimensional structure to be maintained, while providing enough efficiency to be useful as a sensor. Our research efforts focus on developing a technique to make inexpensive, reproducible gratings that are easy to fabricate. By chemically modifying the surface of a commercial grating with a suitable release agent, it is possible to emboss replica gratings onto a variety of waveguide types. The fabrication of embossed gratings will be described, and their performance on glass, ion-diffused, polymer, and semiconductor waveguides will be presented.

Measuring estrogens using flow injection immunoanalysis with liposome amplification

A solid-phase competitive immunoassay is performed in flow injection analysis for the measurement of the hormone 17-beta-estradiol. The flow injection analysis system incorporates a column-type reactor packed with solid silica particles onto which we have covalently immobilized the antigen 17-beta-estradiol. Anti-estradiol is noncovalently conjugated to the liposome through a streptavidin-biotin linkage. When mixed with a sample containing the antigen, the antibody binding sites on the liposomes are complexed which reduces the binding of liposomes to the solid support in a concentration-dependent manner. Sequential immunoassays are performed on-column following a simple regeneration step.

Planar waveguide ion-selective sensors

Abstract A potassium ion-selective planar waveguide total internal reflection (TIR) mode optical sensor was prepared for flow injection application by casting thin film optode membranes onto a glass substrate. For this sensor, a potassium selective ionophore is incorporated in plasticized polyvinyl chloride film sensitized with a hydrogen selective ionophore. The sensor response to potassium ion is determined by measuring the attenuation of a HeNe laser propagated in this thin film device. The laser was coupled into and out of the sensor using grating couplers providing an inexpensive, environmentally stable sensor. The sensitivity of the waveguide based flow injection device is approximately 0.05 mM K + , while the rate of analysis is about 180 samples per hour.

An automated protocol for performance benchmarking a widefield fluorescence microscope

Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other imaging applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy‐to‐use benchmarking methods would facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and would aid the determination of instrumental method validation for commercial product development applications. We describe and evaluate an automated method to characterize a fluorescence imaging systems performance by benchmarking the detection threshold, saturation, and linear dynamic range to a reference material. The benchmarking procedure is demonstrated using two different materials as the reference material, uranyl‐ion‐doped glass and Schott 475 GG filter glass. Both are suitable candidate reference materials that are homogeneously fluorescent and highly photostable, and the Schott 475 GG filter glass is currently commercially available. In addition to benchmarking the analytical performance, we also demonstrate that the reference materials provide for accurate day to day intensity calibration. Published 2014 Wiley Periodicals Inc.

Automated Spectral Smoothing with Spatially Adaptive Penalized Least Squares

A variety of data smoothing techniques exist to address the issue of noise in spectroscopic data. The vast majority, however, require parameter specification by a knowledgeable user, which is typically accomplished by trial and error. In most situations, optimized parameters represent a compromise between noise reduction and signal preservation. In this work, we demonstrate a nonparametric regression approach to spectral smoothing using a spatially adaptive penalized least squares (SAPLS) approach. An iterative optimization procedure is employed that permits gradual flexibility in the smooth fit when statistically significant trends based on multiscale statistics assuming white Gaussian noise are detected. With an estimate of the noise level in the spectrum the procedure is fully automatic with a specified confidence level for the statistics. Potential application to the heteroscedastic noise case is also demonstrated. Performance was assessed in simulations conducted on several synthetic spectra using traditional error measures as well as comparisons of local extrema in the resulting smoothed signals to those in the true spectra. For the simulated spectra, a best case comparison with the Savitzky–Golay smoothing via an exhaustive parameter search was performed while the SAPLS method was assessed for automated application. The application to several dissimilar experimentally obtained Raman spectra is also presented.

Thermal detection of enzyme-labelled antigen-antibody complexes using fiber-optic interferometry

Abstract A fiber-optic interferometer is described for measuring the heat produced in an enzymatic reaction that is localized on the surface of the sensing fiber in a two-arm interferometer. Reactants are introduced to the sensing arm of the interferometer using a flow injection analysis system, while the reference arm is isolated in a separate compartment. Solution concentrations of hydrogen peroxide are quantified using the enzyme catalase immobilized to the sensing fiber. Catalase is also used as a detectable label in a solid-phase thermal immunosensing scheme. These initial results are presented, as well as a discussion of the potential advantages of using a sensor for thermal immunoassays.

Measurement of Scattering and Absorption Cross Sections of Microspheres for Wavelengths between 240 nm and 800 nm.

A commercial spectrometer with a 150 mm integrating sphere (IS) detector was used to estimate the scattering and absorption cross sections of monodisperse polystyrene microspheres suspended in water. Absorbance measurements were performed with the sample placed inside the IS detector. The styrene absorption was non zero for wavelengths less than 300 nm. Correction for fluorescence emission by styrene was carried out and the imaginary part of the index of refraction, ni, was obtained. Absorbance measurements with the sample placed outside the IS detector were sensitive to the loss of photons from the incident beam due to scattering. The absorbance data was fitted with Lorenz-Mie scattering cross section and a correction for the finite acceptance aperture of the spectrometer. The fit parameters were the diameter, the suspension concentration, and the real part of the index of refraction. The real part of the index was parameterized using an expansion in terms of powers of the inverse wavelength. The fits were excellent from 300 nm to 800 nm. By including the imaginary part obtained from the absorbance measurements below 300 nm, it was possible to obtain a good fit to the observed absorbance data over the region 240 nm to 800 nm. The value of ni at 266 nm was about 0.0060±0.0016 for microspheres with diameters of 1.5 μm, 2.0 μm, and 3.0 μm. The scattering cross section, absorption cross section, and the quantum yield at 266 nm of microsphere with a diameter of 2.0 μm was 5.65±0.01 μm2, 1.54±0.03 μm2, and 0.027±0.002 respectively. The styrene absorption reduces the scattering cross section by 20 % at 266 nm.

Standard Reference Material 2036 Near-Infrared Reflection Wavelength Standard

Standard Reference Material 2036 (SRM 2036) is a certified transfer standard intended for the verification and calibration of the wavelength/wavenumber scale of near-infrared (NIR) spectrometers operating in diffuse or trans-reflectance mode. SRM 2036 Near-Infrared Wavelength/Wavenumber Reflection Standard is a combination of a rare earth oxide glass of a composition similar to that of SRM 2035 Near-Infrared Transmission Wavelength/Wavenumber Standard and SRM 2065 Ultraviolet–Visible–Near-Infrared Transmission Wavelength/Wavenumber Standard, but is in physical contact with a piece of sintered poly(tetrafluoroethylene) (PTFE). The combination of glass contacted with a nearly ideal diffusely reflecting backing provides reflection–absorption bands that range from 15% R to 40% R. SRM 2036 is certified for the 10% band fraction air wavelength centroid location, 10%B, of seven bands spanning the spectral region from 975 nm to 1946 nm. It is also certified for the vacuum wavenumber 10%B of the same seven bands in the spectral region from 10 300 cm−1 to 5130 cm−1 at 8 cm−1 resolution. Informational values are provided for the locations of thirteen additional bands from 334 nm to 804 nm.