DIGITAL NIST: An examination of the obstacles and opportunities in the digital transformation of NIST's reference materials.

Early in 2022, NIST embarked on a pilot project to produce digital calibration reports and digital certificates of analysis for reference materials. The goal is to produce examples of digital reports and certificates to assess the scope and challenges of digital transformation in those particular measurement services. This paper focuses on the Reference Material Certificate effort of the pilot project. Our aims for this part of the pilot project are: to generate a digital Reference Material Certificate from certification data; descriptive information about the material, and other data and metadata as needed; to generate a human-readable report from the digital Reference Material Certificate; and to hold a workshop to gather stakeholder feedback. The challenges for NIST include the diverse and complex information presently contained in NIST certificates, converting values to non-SI units to match the needs of stakeholders, and format updates to NIST Reference Material Certificates necessary to allow for machine generation. Other practical challenges include the wide variety of Reference Materials offered by NIST, as well as the needs of internal and external stakeholders. This presentation will report on the progress of the NIST effort and discuss some of the challenges and solutions to producing Digital Reference Material Certificates.

NIST Reference Materials: Utility and Future.

The National Institute of Standards and Technology (NIST), formerly the National Bureau of Standards, was established by the US Congress in 1901 and charged with establishing a measurement foundation to facilitate US and international commerce. This broad language provides NIST with the ability to establish and implement its programs in response to changes in national needs and priorities. This review traces some of the changes in NIST's reference material programs over time and presents the NIST Material Measurement Laboratory's current approach to promoting accuracy and metrological traceability of chemical measurements and validation of chemical measurement processes.

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 system's 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.

Measurement of Microsphere Concentration Using a Flow Cytometer with Volumetric Sample Delivery.

Microsphere concentrations are needed to assign equivalent reference fluorophores (ERF) units to microspheres used in quantitative flow cytometry. A flow cytometer with a syringe based sample delivery system was evaluated for the measurement of the concentration of microspheres contained in a vial of lyophilized microspheres certified by BD Biosciences to contain 50,600 microspheres. The concentration was measured by counting the number of microspheres contained in the volume delivered by the flow cytometer and dividing the number by the volume. The syringe volume was calibrated both in the delivery and draw modes, and the results of the volume calibration were summarized by two calibration lines. The delivered volume was obtained by dividing the number of recorded events by the concentration of microsphere count standard in the sample tube. The draw volume was obtained by weighting the sample tube before and after the draw. The slope of the draw volume calibration line was equal to 1.00 with an offset of -13 µL. The slope of the delivered volume calibration was 0.93 suggesting a systematic volume-dependent bias, which can be rationalized as an effect of suspension flow in capillaries. When the sample volume was set to values between 150 µL and 300 µL, both calibration curves gave similar results suggesting that a good estimate of the true delivered volume can be obtained by subtracting 13 µL from the delivered volume indicated by the syringe settings. The number of microspheres in the volume was obtained by passing the suspension contained in the volume through a laser beam and counting the number of events in which the signals from the scattering and fluorescence detectors exceeded threshold values. Measurements were performed with the lyophilized microspheres made by BD Biosciences and fluorescein microspheres (expired reference material RM 8640) in three buffers: a phosphate buffer saline (PBS), a buffer containing PBS and 0.05 % BSA (bovine serum albumin) by mass, and a buffer containing PBS and 0.05 % TWEEN 20 detergent solution (P1379 Sigma-Aldrich) by mass. It was found that the concentration of count standard was significantly higher in the PBS+BSA buffer relative to the value obtained in PBS buffer. Values for PBS+0.05 % TWEEN 20 buffer were intermediate. The effect of buffer on the measured microsphere concentration was reported previously. The suggested procedure for the measurement of the concentration of microspheres with the flow cytometer is to use PBS+0.05 % BSA buffer, accumulate data for a delivered volume of 150 µL to 300 µL, and reduce the indicated delivered volume by 13 µL when performing the concentration calculation. The procedure was tested on a mixture of lyophilized microspheres and RM 8640 microspheres. The resulting lyophilized microsphere concentration was consistent with the certified value. The RM 8640 concentration determined using the suggested procedure was consistent with the concentration value determined using the relative method with the lyophilized microspheres as the reference. The uncertainties, obtained from one standard deviation of repeated measurements, were about 4 %.

Temperature measurement and optical path-length bias improvement modifications to National Institute of Standards and Technology ozone reference standards.

UNLABELLED: Ambient ozone measurements in the United States and many other countries are traceable to a National Institute of Standards and Technology Standard Reference Photometer (NIST SRP). The NIST SRP serves as the highest level ozone reference standard in the United States, with NIST SRPs located at NIST and at many U.S. Environmental Protection Agency (EPA) laboratories. The International Bureau of Weights and Measures (BIPM) maintains a NIST SRP as the reference standard for international measurement comparability through the International Committee of Weights and Measures (CIPM). In total, there are currently NIST SRPs located in 20 countries for use as an ozone reference standard. A detailed examination of the NIST SRP by the BIPM and NIST has revealed a temperature gradient and optical path-length bias inherent in all NIST SRPs. A temperature gradient along the absorption cells causes incorrect temperature measurements by as much as 2 degrees C. Additionally, the temperature probe used for temperature measurements was found to inaccurately measure the temperature of the sample gas due to a self-heating effect. Multiple internal reflections within the absorption cells produce an actual path length longer than the measured fixed length used in the calculations for ozone mole fractions. Reflections from optical filters located at the exit of the absorption cells add to this effect. Because all NIST SRPs are essentially identical, the temperature and path-length biases exist on all units by varying amounts dependent upon instrument settings, laboratory conditions, and absorption cell window alignment. This paper will discuss the cause of and physical modifications for reducing these measurement biases in NIST SRPs. Results from actual NIST SRP bias upgrades quantifying the effects of these measurement biases on ozone measurements are summarized. IMPLICATIONS: NIST SRPs are maintained in laboratories around the world underpinning ozone measurement calibration and traceability within and between countries. The work described in this paper quantifies and shows the reduction of instrument biases in NIST SRPs improving their overall agreement. This improved agreement in all NIST SRPs provides a more stable baseline for ozone measurements worldwide.

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 µm(2), 1.54±0.03 µm(2), and 0.027±0.002 respectively. The styrene absorption reduces the scattering cross section by 20 % at 266 nm.

Measurement of Scattering and Absorption Cross Sections of Dyed Microspheres.

Measurements of absorbance and fluorescence emission were carried out on aqueous suspensions of polystyrene (PS) microspheres with a diameter of 2.5 µm using a spectrophotometer with an integrating sphere detector. The apparatus and the principles of measurements were described in our earlier publications. Microspheres with and without green BODIPY(@) dye were measured. Placing the suspension inside an integrating sphere (IS) detector of the spectrophotometer yielded (after a correction for fluorescence emission) the absorbance (called A in the text) due to absorption by BODIPY(@) dye inside the microsphere. An estimate of the absorbance due to scattering alone was obtained by subtracting the corrected BODIPY(@) dye absorbance (A) from the measured absorbance of a suspension placed outside the IS detector (called A1 in the text). The absorption of the BODIPY(@) dye inside the microsphere was analyzed using an imaginary index of refraction parameterized with three Gaussian-Lorentz functions. The Kramer-Kronig relation was used to estimate the contribution of the BODIPY(@) dye to the real part of the microsphere index of refraction. The complex index of refraction, obtained from the analysis of A, was used to analyze the absorbance due to scattering ((A1 - A) in the text). In practice, the analysis of the scattering absorbance, A1-A, and the absorbance, A, was carried out in an iterative manner. It was assumed that A depended primarily on the imaginary part of the microsphere index of refraction with the other parameters playing a secondary role. Therefore A was first analyzed using values of the other parameters obtained from a fit to the absorbance due to scattering, A1-A, with the imaginary part neglected. The imaginary part obtained from the analysis of A was then used to reanalyze A1-A, and obtain better estimates of the other parameters. After a few iterations, consistent estimates were obtained of the scattering and absorption cross sections in the wavelength region 300 nm to 800 nm.

Measurement of Scattering Cross Section with a Spectrophotometer with an Integrating Sphere Detector.

A commercial spectrometer with an integrating sphere (IS) detector was used to measure the scattering cross section of microspheres. Analysis of the measurement process showed that two measurements of the absorbance, one with the cuvette placed in the normal spectrometer position, and the second with the cuvette placed inside the IS, provided enough information to separate the contributions from scattering and molecular absorption. Measurements were carried out with microspheres with different diameters. The data was fitted with a model consisting of the difference of two terms. The first term was the Lorenz-Mie (L-M) cross section which modeled the total absorbance due to scattering. The second term was the integral of the L-M differential cross section over the detector acceptance angle. The second term estimated the amount of forward scattered light that entered the detector. A wavelength dependent index of refraction was used in the model. The agreement between the model and the data was good between 300 nm and 800 nm. The fits provided values for the microsphere diameter, the concentration, and the wavelength dependent index of refraction. For wavelengths less than 300 nm, the scattering cross section had significant spectral structure which was inversely related to the molecular absorption. This work addresses the measurement and interpretation of the scattering cross section for wavelengths between 300 nm and 800 nm.

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.

Use of Standard Reference Material 2242 (Relative Intensity Correction Standard for Raman Spectroscopy) for microarray scanner qualification.

As a critical component of any microarray experiment, scanner performance has the potential to contribute variability and bias, the magnitude of which is usually not quantified. Using Standard Reference Material (SRM) 2,242, which is certified for Raman spectral correction, for monitoring the microarray fluorescence at the two most commonly used wavelengths, our team at the National Institute of Standards and Technology (NIST) has developed a method to establish scanner performance, qualifying signal measurement in microarray experiments. SRM 2,242 exhibits the necessary photostability at the excitation wavelengths of 635 nm and 532 nm, which allows scanner signal stability monitoring, although it is not certified for use in this capacity. In the current study, instrument response was tracked day to day, confirming that changes observed in experimental arrays scanned are not due to changes in the scanner response. Signal intensity and signal-to-noise ratio (S/N) were tracked over time on three different scanners, indicating the utility of the SRM for scanner qualification.

Requirements for relative intensity correction of Raman spectra obtained by column-summing charge-coupled device data.

The relative intensity correction of Raman spectra requires the measurement of a source of known relative irradiance. Raman spectrometers that employ two-dimensional charge-coupled device (CCD) array detectors may be operated in two distinct modes. One mode directly measures the counts in each CCD pixel, but more commonly for the collection of spectra, the counts in the CCD row pixels are summed for a given column. If distortions in the corrected spectral shapes are to be avoided, operation in the mode where rows are summed places restrictions on the spatial intensity profile of the source of known irradiance that is used for the relative intensity correction procedure and, in some cases, also on the spatial intensity profile of the measured Raman light. Numerical expressions are given from which these restrictions can be derived. Magnitudes of distortions that can arise when intensity-correcting spectra obtained with CCD data where rows in a column are summed are estimated by modeling different cases. Data are given showing the inherent pixel quantum efficiency variation that exists in CCDs. Spectra are given showing the effects of a local area of significant change in pixel quantum efficiency that was found to be present on one CCD detector.

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.

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.

Rare-earth glass reference materials for near-infrared spectrometry: correcting and exploiting temperature dependencies.

Quantitative descriptions of the location of seven near-infrared absorption bands as functions of temperature 5-50 degrees C are presented here for three recently introduced wavelength/wavenumber Standard Reference Materials (SRMs): SRM 2035, SRM 2065, and SRM 2036. For all bands in all three SRMs, locations are well described as linear models parametrized with the location at 0 degrees C (intercept) and the rate of location change per degrees C (slope). Since these materials were produced from compositionally similar melts, the slopes for each band are identical within measurement imprecision in all three SRMs; only minor differences are observed in the intercepts. Because the direction of change in location differs among the bands, it is possible to use the measured band locations to reliably estimate sample temperature. Two approaches to estimating temperature are evaluated: slope and measurement uncertainty-weighted means. While both methods work well with measurements made under well-characterized and stable environmental conditions, the more complex uncertainty-weighted analysis becomes relatively more predictive as the total measurement uncertainties increase.