Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges.

Cross-Calibration of an α-Source Used for Luminescence Dating by Applying Different Samples and Procedures

In terms of fine-grain luminescence dating applications, the efficiency of α-radiation in producing luminescence is an important issue when determining environmental dose rates. Efficiency is usually assessed by measuring the ratio of luminescence intensities induced by known α and β laboratory doses. Consequently, most thermoluminescence (TL)/optically stimulated luminescence (OSL) readers besides the standard 90Sr/90Y β-source can also be equipped with a 241Am α-source. A crucial point is, however, the calibration of these sources. The calibration of β-sources is routinely performed using standard quartz samples previously irradiated by a known γ-dose, though, in the case of α-sources, the procedure is less standardised, partly because there are no calibration materials with a known α-efficiency value. In this study, we aimed to cross-calibrate the built-in α-source of a RISØ TL/OSL DA-20 luminescence reader by testing and comparing five procedures, applying different samples (quartz and polymineral), different protocols multiple aliquot regeneration (MAR) and single aliquot regeneration (SAR) and different calibration sources. Throughout the tests, the performance of the fine-grain RISØ calibration quartz was also assessed. Regardless of the applied procedure, the calculated α-dose rates with one exception gave similar results. On the one hand, the applied polymineral sample due to potential fading, fairly high residuals after bleaching and relatively low infrared stimulated luminescence (IRSL) sensitivity proved to be the least optimal choice for cross-calibration. On the other hand, the tested natural fine grain quartz gave almost identical results when using different types of bleaching and different calibration α-sources. The mean dose rate determined for the source was 0.080 ± 0.004 Gy/s. The cross-calibration by using the RISØ fine grain quartz yielded somewhat higher but at the apparent uncertainty of luminescence dating still not significantly different dose rate for the source under calibration. Tests showed that the calibration quartz saturates at a relatively low α-dose, and the shape of α- and β-dose-response curves also depart from each other quite early, suggesting that cross-calibration with this material seems to be reliable only at low doses. For the first time, the a-value of the fine-grain calibration quartz was also determined using the freshly calibrated α-source, and the measurement yielded a 0.054 ± 0.003 value. We propose that after further validation of this result, the RISØ calibration quartz can ease the dose rate assessment of uncalibrated α-sources in the future.

Calibration of HVI cotton elongation measurements

Background The strength of cotton fiber has been extensively studied and significant improvements in fiber strength have been made, but fiber elongation has largely been ignored, despite it contributing to the energy needed to break fibers, which affects fiber handling and processing. High Volume Instruments (HVI) measure fiber elongation but have not been calibrated for this property, making the measurement unavailable for comparative work among instruments. In prior work, a set of elongation calibration materials had been developed based on Stelometer results. A round trial of ten Australian and U.S. instruments was conducted on six cotton samples representing a range of 4.9% to 8.1% elongation. Results By scaling the HVI elongation values of each instrument to the values of the two calibration samples, the coefficient of variation in instrument measurements was reduced from an average of 34% for the uncalibrated measurements to 5% for the calibrated measurements. The reduction in variance allows for the direct comparison of results among instruments. A single-point elongation calibration was also assessed but found to be less effective than the proposed two-point calibration. Conclusion The use of an effective calibration routine on HVI measurement of cotton significantly reduces the coefficient of variation of the elongation measurement within and between instruments. The implementation of the elongation calibration will allow testing and breeding programs to implement high-speed elongation testing which makes the use of elongation values possible in breeding programs.

Arsenic and Lead Determination in D&C Red No. 6 Lakes and D&C Red No. 7 Lakes Containing Barium Sulfate Using X-Ray Fluorescence Spectrometry

Background Color additives requiring batch certification by the U.S. Food and Drug Administration have Code of Federal Regulations (CFR) specification limits for certain elements and are usually analyzed by x-ray fluorescence spectrometry (XRF). However, analysis of some color additives presents difficulties. Objective An XRF method was developed for quickly determining whether barium sulfate-containing color additives certifiable as D&C Red No. 6 lakes and D&C Red No. 7 lakes meet the CFR specifications for arsenic (As) and lead (Pb). Method Difficulties in preparing XRF standards and analyzing As and Pb in matrices with the heavy x-ray absorber barium (Ba) were overcome by first preparing As- and Pb-fortified cellulose, then blending color additive samples with fortified and unfortified cellulose to produce XRF calibration materials. Satisfactory compensation for changes in intensity caused by Ba absorption was achieved by measuring varying concentrations of As and Pb at several concentrations of Ba. Results Test samples of barium sulfate-containing D&C Red No. 6 lakes and D&C Red No. 7 lakes were analyzed by the XRF method and by an inductively coupled plasma-mass spectrometry (ICP-MS) method. Conclusions The ICP-MS method poses some difficulties and is time consuming. In contrast, the XRF method requires very little sample preparation, is nondestructive, uses calibrations that are stable for long periods of time, and offers acceptable determination limits (1 mg/kg As, 4 mg/kg Pb) which are less than the CFR specification limits (3 mg/kg As, 20 mg/kg Pb). Highlights The new XRF method is applicable for use in routine batch certification.

Establishing SI-Traceability of Nanoparticle Size Values Measured with Line-Start Incremental Centrifugal Liquid Sedimentation

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful technique to determine particle size based on the principles of Stokes’ law. As most input quantities of the Stokes equation cannot be easily determined for typical instruments used for this method, an alternative method which depends on calibrating the sedimentation time scale with reference particles has become common practice. Unfortunately, most of these calibration materials (calibrants) come with limited information regarding their metrological reliability (e.g., lack of measurement uncertainties and traceability statements, incomplete measurand definitions). As a consequence, routine particle size results obtained by disc-CLS are mostly only traceable to the calibrant used, and effective comparisons can only be made for those results originating from measurements performed with the same types of calibrants. In this study, we discuss the concept of metrological traceability and demonstrate that particle size results obtained by disc-CLS can be traceable to the ultimate metrological reference, i.e., the unit of length in the International System of Units (SI), the meter. Using the example of two colloidal silica certified reference materials, we describe how laboratories can realize metrological traceability to the SI by simplifying complex traceability networks.

Technical note: An empirical method for absolute calibration of coccolith thickness

As major calcifiers in the open ocean, coccolithophores play a key role in the marine carbon cycle. Because they may be sensitive to changing CO2 and ocean acidification, there is significant interest in quantifying past and present variations in their cellular calcification by quantifying the thickness of the coccoliths or calcite plates that cover their cells. Polarized light microscopy has emerged as a key tool for quantifying the thickness of these calcite plates, but the reproducibility and accuracy of such determinations has been limited by the absence of suitable calibration materials in the thickness range of coccoliths (0–4 µm). Here, we describe the fabrication of a calcite wedge with a constant slope over this thickness range, and the independent determination of calcite thickness along the wedge profile. We show how the calcite wedge provides more robust calibrations in the 0 to 1.55 µm range than previous approaches using rhabdoliths. We show the particular advantages of the calcite wedge approach for developing equations to relate thickness to the interference colors that arise in calcite in the thickness range between 1.55 and 4 µm. The calcite wedge approach can be applied to develop equations relevant to the particular light spectra and intensity of any polarized light microscope system and could significantly improve inter-laboratory data comparability.

An empirical method for absolute calibration of coccolith thickness

As major calcifiers in the open ocean, coccolithophores play a key role in the marine carbon cycle. Because they may be sensitive to changing CO2 and ocean acidification, there is significant interest in quantifying past and present variations in their cellular calcification by quantifying the thickness of the coccoliths or calcite plates that cover their cells. Polarized light microscopy has emerged as a key tool for quantifying the thickness of these calcite plates, but the reproducibility and accuracy of such determinations has been limited by the absence of suitable calibration materials in the thickness range of coccoliths (0–4 microns). Here, we describe the fabrication of a calcite wedge with a constant slope over 15 this thickness range, and the independent determination of calcite thickness along the wedge profile. We show how the calcite wedge provides more robust calibrations in the 0 to 1.55 μm range than previous approaches using rhabdoliths. We show the particular advantages of the calcite wedge approach for developing equations to relate thickness to the interference colors that arise in calcite in the thickness range between 1.55 and 4 μm. The calcite wedge approach can be applied to develop equations relevant to the particular light spectra and intensity of any polarized light microscope system and could significantly improve within and inter-laboratory data comparability.

Influence of calibration method and material on the accuracy of stress distribution measurement systems

Biomechanical analyses of the stress distribution and the force transfer in the human knee are essential to better understand the aetiology of joint diseases. Accuracy studies of commonly used capacitive or resistive-based stress distribution measurement systems have led to severe problems caused by an inaccurate experimental setup. For instance, in one study, overestimations of the measured forces in the sensor’s centre were reported. Therefore, the primary aim of this study was to investigate the ability of capacitive and resistive-based sensors to measure forces in a homogenous pressure environment and the secondary goal was to analyse the influence of different calibration materials on the measurement accuracy. A Novel pressure vessel and metal indenters covered with different rubber materials were used in combination with a material testing machine to load the sensors. Four different linearly increasing nominal forces (925–3670 N) were applied and the deviations between the nominal and the measured forces were calculated. The capacitive measurement system showed errors between 1% and 7% in the homogenous pressure environment, whereas the errors of the resistive system were found to vary between 4% and 17%. The influence of the calibration material was observed to be greater for the resistive sensors (1–179%) than for the capacitive sensors (0.5–25%). In conclusion, it can be stated that – for the pressure measurement systems compared in this article – the capacitive one is less sensitive to the calibration method and the calibration material than the resistive system.