Fluorescence Calibration Research Articles

Alamar Blue assay optimization to minimize drug interference and inter assay viability

Alamar Blue (AB) has become an increasingly popular reagent of choice for cell viability assays. We chose AB over other reagents such as MTT and Cell-Titer Glo due to its cost effectiveness and its ability to be a nondestructive assay. While analyzing the effect of osimertinib, an EGFR inhibitor on the non-small cell lung cancer cell line, PC-9, we noticed unexpected right-shifts of dose response curves as compared to the curves obtained by Cell Titer Glo assay. Here, we describe our modified AB assay method to avoid that right shift in dose response curves.•Removal of the drug containing medium prior to AB addition eliminated the falsely increased readings, giving comparable dose response curves as determined by Cell Titer Glo assay.•Plate-to-plate variability can be minimized by adding an appropriate concentration of a common fluorescence calibration standard to the assay plates to calibrate fluorimeter sensitivity.•Alamar Blue can be used as a continuous longitudinal assay to monitor cell growth or recovery from drug toxicity over time.

Practices and Rules of 16th Century Genoese Gilding: Exploring Gold Leaf Thickness and Caratage through X-ray and Ion Beam Techniques

This study investigates the practices and rules of Genoese gilding, drawing insights from a 16th-century manuscript containing regulations for gold leaf production. Employing X-ray and ion beam techniques, we quantitatively assess the manuscript’s gold leaf thickness without destructive sampling. Artisanal goldbeater-produced leaves of different thicknesses, applied with a guazzo or mordant technique, served as standards. Further analysis of samples with unknown thickness from the furniture of Palazzo Spinola di Pellicceria in Genoa (Italy) has confirmed the method’s applicability to practical cases. External beam Rutherford backscattering spectrometry (RBS) and particle-induced X-ray emission (PIXE) analyses were carried out using 3 MeV protons at the LABEC accelerator laboratory in Florence. A linear relationship between Gold Lα peak yield and leaf thickness, as measured by RBS, has been established for optimal calibration of portable or hand-held X-Ray fluorescence (XRF) instrumentation for in situ measurements. Moreover, the caratage of the gold foil preserved in the manuscript has been assessed.

Portable fiber-optic sensor for simple, fast, cost-effective, and environmentally friendly quantification of total acidity in real-world applications

In this work, an innovative fiber-optic sensor for measuring total acidity (TA: expressed as the % w/v of acetic acid) in beverages was developed. For this purpose, we relied on the kinetic principles of heterogeneous solid-phase reactions (slow reactions due to diffusion processes) to develop a novel pH-sensitive fluorescent membrane calibration method for the quantification of TA. In the proposed calibration method, a linear relationship between TA and the protonation rate of a pH-sensitive fluorescent membrane with a pKa ≥ 9 (Paper-FM) is established. Paper-FM was synthesized by the covalent immobilization of the pH-probe Nile Blue on a porous paper membrane. In addition, a 3D-printed interface was designed to implement Paper-FM into a 0.5 mm diameter optical fiber. The fiber-optic sensor developed in this work was tested by analyzing 21 vinegar samples of different origins, and the results were successfully validated with a reference titration method. The results presented in this work show that our fiber-optic sensor allows a direct, simple, and fast (15 s response time) quantification of TA in vinegar samples. In addition, it is environmentally friendly, because, unlike titration methods, it does not generate toxic waste, is reversible, and is extremely stable (a 0.9 mm diameter circle of Paper-FM allows at least 50 consecutive measurements).

Rapid, label-free, contactless measurement of membrane potential in excitable H9c2 cardiomyoblasts using ζ-potential

The measurement of cell membrane potential (V m) is important for understanding ion channel function. V m plays a role in several routine cellular functions and diseases, particularly in excitable cells such as muscle and nerve. However, measuring V m is difficult, relying either on labour-intensive direct measurement of single cells (intracellular electrodes, patch clamp) or indirect measurement of fluorescence intensity, using V m-sensitive labels. Here we demonstrate a direct measurement technique based on determination of the cell’s ζ-potential, the electrical potential at the hydrodynamic shear plane, approximately 1 nm beyond the cell surface. We demonstrate this principle using excitable H9c2 cardiomyoblasts, measured in both polarised and depolarised states, before and after extracellular intervention to alter cell ion concentration. Given widespread availability of ζ-potential measurement apparatus (most typically in chemistry and materials science settings), this offers a new method of measuring V m without the need for fluorescence measurements or calibration curves.

Measuring differential fitness costs and interactions between genetic cassettes using fluorescent spectrophotometry.

In this article, we present a method for designing, executing, and analyzing data from a microbial competition experiment. We use fluorescent reporters to label different competing strains and resolve individual growth curves using a fluorescent spectrophotometer. Our comprehensive data analysis pipeline integrates multiple experiments to simultaneously infer sources of variation, extract selection coefficients, and estimate the genetic contributions to fitness for various synthetic genetic cassettes (SGCs). To demonstrate the method, we employ a synthetic biological system based on Escherichia coli. Strains carry 1 of 10 different plasmids and one of three genomically integrated fluorescent markers. All strains are co-cultured to obtain real-time measurements of optical density (total population density) and fluorescence (sub-population densities). We identify challenges in calibrating between fluorescence and density and of fluorescent proteins maturing at different rates. To resolve these issues, we compare two methods of fluorescence calibration and correct for maturation by measuring in vivo maturation times. We provide evidence of genetic interactions occurring between our SGCs and further show how to use our statistical model to test some hypotheses about microbial growth and the costs of protein expression.IMPORTANCEFluorescently labeled co-cultures are becoming increasingly popular. The approach proposed here offers a high standard for experimental design and data analysis to measure selection coefficients and growth rates in competition. Measuring competitive differences is useful in many laboratory studies, allowing for fitness cost-correction of growth rates and ecological interactions and testing hypotheses in synthetic biology. Using time-resolved growth curves, rather than endpoint measurements, for competition assays allows us to construct a detailed scientific model that can be used to ask questions about fine-grained phenomena, such as bacterial growth dynamics, as well as higher-level phenomena, such as the interactions between synthetic cassette expression.

Reflection transformation calibration for mirror-assisted multi-view digital image correlation system using fluorescent speckle patterns

Mirror-assisted multi-view digital image correlation (MV-DIC) is a simple-to-implement DIC technique for panoramic kinematic field measurements of regular-sized specimens. In the practical implementation of mirror-assisted MV-DIC, accurate reflection transformation calibration of the planar mirrors is considered as a pivotal step. In this work, we propose to use fluorescent speckle patterns fabricated on the mirrors to calibrate their reflection transformation matrices, which can fully utilize the camera’s spatial resolution and simplify the calibration processes. The fluorescent speckle patterns on the mirrors do not interfere with the virtual object surfaces using a regular blue-light stereo-DIC system. However, under the illumination of ultraviolet light, the fluorescent speckle patterns can be captured and used to calibrate the position and orientation of the planar mirrors. The practicality and efficacy of the fluorescent speckle pattern-based calibration method are validated by profile measurement and translation tests, and an application in a four-point bending experiment is also demonstrated.

Efficiency scale for scattering luminescent particles linked to fundamental and measurable spectroscopic properties

Comparing the performance of molecular and nanoscale luminophores and luminescent micro- and nanoparticles and estimating achievable signal amplitudes and limits of detection requires a standardizable intensity scale. This initiated the development of the relative MESF (number of molecules of equivalent soluble fluorochromes) and ERF (equivalent reference fluorophores) scales for flow cytometry and fluorescence microscopy. Both intensity scales rely on fluorescence intensity values assigned to fluorescent calibration beads by an intensity comparison to spectrally closely matching fluorophore solutions of known concentration using a spectrofluorometer. Alternatively, the luminophore or bead brightness (B) can be determined that equals the product of the absorption cross section (σa) at the excitation wavelength (σa(λex)) and the photoluminescence quantum yield (Φpl). Thereby, an absolute scale based on fundamental and measurable spectroscopic properties can be realized which is independent of particle size, material, and luminophore staining or labeling density and considers the sensitivity of the optical properties of luminophores to their environment. Aiming for establishing such a brightness scale for light-scattering dispersions of luminescent particles with sizes exceeding a few ten nanometers, we demonstrate how the brightness of quasi-monodisperse 25 nm, 100 nm, and 1 µm sized polystyrene particles (PSP), loaded with two different dyes in varying concentrations, can be obtained with a single custom-designed integrating sphere setup that enables the absolute determination of Φpl and transmittance and diffuse reflectance measurements. The resulting Φpl, σa(λex), imaginary parts of the refractive index, and calculated B values of these samples are given in dependence of the number of incorporated dye molecule per particle. Finally, a unitless luminescence efficiency (LE) is defined allowing for the direct comparison of luminescence efficiencies of particles with different sizes.

Fluholoscopy—Compact and Simple Platform Combining Fluorescence and Holographic Microscopy

The combination of different imaging modalities into single imaging platforms has a strong potential in biomedical sciences as it permits the analysis of complementary properties of the target sample. Here, we report on an extremely simple, cost-effective, and compact microscope platform for achieving simultaneous fluorescence and quantitative phase imaging modes with the capability of working in a single snapshot. It is based on the use of a single illumination wavelength to both excite the sample's fluorescence and provide coherent illumination for phase imaging. After passing the microscope layout, the two imaging paths are separated using a bandpass filter, and the two imaging modes are simultaneously obtained using two digital cameras. We first present calibration and analysis of both fluorescence and phase imaging modalities working independently and, later on, experimental validation for the proposed common-path dual-mode imaging platform considering static (resolution test targets, fluorescent micro-beads, and water-suspended lab-made cultures) as well as dynamic (flowing fluorescent beads, human sperm cells, and live specimens from lab-made cultures) samples.

Quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity.

This study presented a quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity. Droplets encapsulating single cells and even distribution of fluorescein labeled antibodies removed from targeted cells deformed through the constriction microchannel where the excited fluorescent signals were sampled and interpreted into numbers of proteins based on volume equivalence in measurement of droplets and calibration of fluorescence. To improve the detection reliability, a comprehensive analysis and comparison of multiple stripping agents such as proteinase K, guanidine hydrochloride, and urea was conducted. To improve the detection sensitivity, light modulation was used to address electrical noises and quartz microchannels were fabricated to address optical noises. As a demonstration, based on this quantitative flow cytometry of droplet microfluidics, (1) mutant p53 expressions of single cells were quantified as 1.95 ± 0.60 × 105 (ncell =2918 of A431) and 1.30 ± 0.70 × 105 (ncell =3954 of T47D); (2) single-cell expressions of Ras, c-Myc, and β-tubulin were quantified as 1.90 ± 0.59 × 105 , 4.39 ± 1.44 × 105 , and 2.97 ± 0.81 × 105 (ncell =3298 of CAL 27), 1.83 ± 0.58 × 105 , 2.08 ± 0.13 × 106 , and 1.96 ± 0.74 × 105 (ncell =5459 of WSU-HN6). As a microfluidic tool capable of quantitatively estimating single-cell protein expressions, this methodology may provide a new quantitative perspective for the field of flow cytometry.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Development of droplet microfluidics capable of quantitative estimation of single-cell multiplex proteins

This study presented constriction microchannel based droplet microfluidics realizing quantitative measurements of multiplex types of single-cell proteins with high throughput. Cell encapsulation with evenly distributed fluorescence labelled antibodies stripped from targeted proteins by proteinase K was injected into the constriction microchannel with the generated fluorescence signals captured and translated into protein numbers leveraging the equivalent detection volume formed by constriction microchannels in both droplet measurements and fluorescence calibration. In order to form the even distribution of fluorescence molecules within each droplet, the stripping effect of proteinase K to decouple binding forces between targeted proteins and fluorescence labelled antibodies was investigated and optimized. Using this microfluidic system, binding sites for beta-actin, alpha-tubulin, and beta-tubulin were measured as 1.15 ± 0.59 × 106, 2.49 ± 1.44 × 105, and 2.16 ± 1.01 × 105 per cell of CAL 27 (N cell = 15 486), 0.98 ± 0.58 × 106, 1.76 ± 1.34 × 105 and 0.74 ± 0.74 × 105 per cell of Hep G2 (N cell = 18 266). Neural net pattern recognition was used to differentiate CAL 27 and Hep G2 cells, producing successful rates of 59.4% (beta-actin), 64.9% (alpha-tubulin), 88.8% (beta-tubulin), and 93.0% in combination, validating the importance of quantifying multiple types of proteins. As a quantitative tool, this approach could provide a new perspective for single-cell proteomic analysis.

Automated analysis of bacterial flow cytometry data with FlowGateNIST.

Flow cytometry is commonly used to evaluate the performance of engineered bacteria. With increasing use of high-throughput experimental methods, there is a need for automated analysis methods for flow cytometry data. Here, we describe FlowGateNIST, a Python package for automated analysis of bacterial flow cytometry data. The main components of FlowGateNIST perform automatic gating to differentiate between cells and background events and then between singlet and multiplet events. FlowGateNIST also includes a method for automatic calibration of fluorescence signals using fluorescence calibration beads. FlowGateNIST is open source and freely available with tutorials and example data to facilitate adoption by users with minimal programming experience.

Biofortification quality in bananas monitored by energy-dispersive X-ray fluorescence and chemometrics

Biofortification is a nutritional strategy used to enhance nutrients in a variety of staple foods. As bananas and plantains (Musa spp.) are considered staple food in many developing countries, monitoring zinc (Zn) content in biofortified bananas is crucial to ensure this mineral intake. Bananas were biofortified by injecting Zn sulfate heptahydrate (ZnSO4·7H2O) solutions into banana trees' pseudostem (1%, 2%, and 4%) compared with the control treatment. Zinc content was estimated using energy-dispersive X-ray fluorescence (EDXRF) and multivariate calibration using partial least squares (PLS). The impressive result is the possibility of high throughput analysis of Zn in bananas after biofortification to guarantee the quality when eaten as a central portion of the diet.

Insights into the inter-element effects in the EDXRF determination of zirconium in binary aqueous solutions via the calibration method

Calibration of the energy dispersive X-ray fluorescence (EDXRF) spectrometer, using pure elemental standard solutions of Cu, Zr, Nb, Mo, Ta, and U, was carried out and the instrumental sensitivity was derived for each. The effects of the presence of the rest of these elements on the calibration of Zr were evaluated by analyzing the respective binary solutions, separately. All the matrix elements led to a reduction in the Zr- sensitivity of the spectrometer. Intercepts of the calibration curves were found to decrease with Cu, Mo. Nb, Ta, and U led to the elevation of the intercepts. The highest specific changes (per mg of the matrix) in both the slope and intercept were observed upon the addition of Mo. Numerically, these values were -5,917 and -1,187 respectively. Limits of detection for Zr, from the calibration curve, were the best (0.28 μg) in the presence of 7.8 mg Mo and the worst (165.4 μg) in the presence of 53 mg U. The results were validated by multiple standard additions on a sample and analysis of reference standard solution of Zr. Quality control sample analysis re-affirmed the accuracy of the present results. Repeatability was also established for the measurements of solution samples.

Fluorescence and colorimetric dual-mode sensor for visual detection of malathion in cabbage based on carbon quantum dots and gold nanoparticles

A dual-mode fluorescence/colorimetric sensor based on carbon quantum dots (CQDs) and gold nanoparticles (GNPs) was developed for visual detection of malathion in cabbage. The CQDs-GNPs nanocomposite exhibited emission wavelength at 527 nm and absorption wavelength at 524 nm. The fluorescence intensity increased and absorption decreased with addition of malathion. Fluorescence and colorimetric calibration curves were established based on fluorescence intensity (R2 = 0.9914) and absorbance (R2 = 0.9608) in the range of 1 × 10−9–1 × 10−2 M, respectively. Furthermore, fluorescence and colorimetric standard arrays were prepared for visual detection of malathion according to the change of fluorescence brightness and color. Finally, the approximate concentrations of malathion in cabbage samples were estimated by the standard arrays and naked eyes. The calibration curves were used for accurate detection in cabbage samples with recoveries of 89.9%–103.4% (fluorescence) and 88.7%–107.6% (colorimetric). The established sensor for visual malathion detection in cabbage was accurate with strong application potential, especially for rapid screening.

Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry.

Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the “tip of the iceberg” of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.

Spectral Standards Based on Glasses Activated with Rare-Earth Element Ions for the Calibration of Fluorescence and Raman Spectrometers

The results of development of fluorescence calibration standards based on phosphate matrix glass activated with ions of rare-earth metals are presented. The calibration standards are designed for spectral correction of emission spectra and systemic monitoring of sensitivity fluctuations in spectrofluorimeters. The possibility of using them for the calibration of Raman spectrometers is considered.

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

Small Particle Fluorescence and Light Scatter Calibration Using FCMPASS Software.

Use of flow cytometry to analyze small particles has been implemented for several decades. More recently, small particle analysis has become increasingly utilized owing to the increased sensitivity of conventional and commercially available flow cytometers along with growing interest in small particles such as extracellular vesicles (EVs). Despite an increase in small particle flow cytometry utilization, a lack of standardization in data reporting has resulted in a growing body of literature regarding EVs that cannot be easily interpreted, validated, or reproduced. Methods for fluorescence and light scatter standardization are well established, and the reagents to perform these analyses are commercially available. Here, we describe FCMPASS , a software package for performing fluorescence and light scatter calibration of small particles while generating standard reports conforming to the MIFlowCyt-EV standard reporting framework. This article covers the workflow of implementing calibration using FCMPASS as follows: acquisition of fluorescence and light scatter calibration materials, cataloguing the reference materials for use in the software, creating cytometer databases and datasets to associate calibration data and fcs files, importing fcs files for calibration, inputting fluorescence calibration parameters, inputting light scatter calibration parameters, and applying the calibration to fcs files. Published 2020. U.S. Government. Basic Protocol 1: Acquisition and gating of light scatter calibration materials Basic Protocol 2: Acquisition and gating of fluorescence calibration materials Alternate Protocol: Cross-calibration of fluorescence reference materials Basic Protocol 3: Cataloguing light scatter calibration materials Basic Protocol 4: Cataloguing fluorescence calibration materials Basic Protocol 5: Creating cytometer databases and datasets Basic Protocol 6: Importing fcs files Basic Protocol 7: Fluorescence calibration Basic Protocol 8: Light scatter calibration Basic Protocol 9: Performing and reporting fcs file calibration.

Sensitivity for Multivariate Calibration Based on Multilayer Perceptron Artificial Neural Networks.

The use of machine learning for multivariate spectroscopic data analysis in applications related to process monitoring has become very popular since non-linearities in the relationship between signal and predicted variables are commonly observed. In this regard, the use of artificial neural networks (ANN) to develop calibration models has demonstrated to be more appropriate and flexible than classical multivariate linear methods. The most frequently reported type of ANN is the so-called multilayer perceptron (MLP). Nevertheless, the latter models still lack a complete statistical characterization in terms of prediction uncertainty, which is an advantage of the parametric counterparts. In the field of analytical calibration, developments regarding the estimation of prediction errors would derive in the calculation of other analytical figures of merit (AFOMs), such as sensitivity, analytical sensitivity, and limits of detection and quantitation. In this work, equations to estimate the sensitivity in MLP-based calibrations were deduced and are here reported for the first time. The reliability of the derived sensitivity parameter was assessed through a set of simulated and experimental data. The results were also applied to a previously reported MLP fluorescence calibration methodology for the biopharmaceutical industry, yielding a value of sensitivity ca. 30 times larger than for the univariate reference method.