Fluorescence Calibration Research Articles

Chemical analysis of argon-oxygen decarburization slags in stainless steelmaking process by X-ray fluorescence spectrometry

A quantitative analysis method of fluorine in the slags produced in the stainless argon–oxygen decarburization (AOD) process by X-ray spectrometry was proposed employing direct analysis technique by pressed pellet. This research investigated the separate quantification of calcium fluoride and calcium oxide contained in AOD slags. X-ray diffraction measurement was performed to identify the stable phase of fluorine compound, which is primarily present in the slags. The synthetic standards prepared in laboratory were used to construct the X-ray fluorescence (XRF) calibration curves for F, CatO, SiO2, MgO, Al2O3 and Cr2O3, considering the matrix effects and line overlap corrections (t: total). The calibration curves were tested by the quantitative analysis of synthetic standards with satisfactory precision and accuracy. The proposed method might be an alternative solution to the problem with the simple and routine chemical analyses of calcium fluoride in AOD slags of stainless steelmaking process by XRF spectrometry. Copyright © 2010 John Wiley & Sons, Ltd.

Non-invasive method to quantify local bacterial concentrations in a mixed culture biofilm

To better engineer and analyze beneficial biofilms as well as to develop strategies to control detrimental biofilms (e.g., biomedical device-based infections), it is critical to quantify bacterial species compositions within biofilms. A non-invasive method is described here that determines local and overall bacterial concentrations within a biofilm, using optical microscopy and digital image analysis techniques. The method is based upon a calibration of cell fluorescence to known cell number concentrations and is verified by direct cell counts of destructive samples of cultivated biofilms. Two GFP mutants, each with unique emission colors were used with both epi-fluorescent microscopy and one-photon confocal microscopy to determine local spatial biofilm cell concentrations in pure and mixed-strain biofilms. Our microbial system comprises Pseudomonas putida containing either green fluorescent protein (GFP) or containing the red fluorescent protein (DsRed). Strains expressing a green or red fluorescent protein were detected by two different microscopy methods: epi-fluorescence and single-photon confocal laser scanning microscopy. Overall biofilm cell concentrations determined directly from destructive samples were in good agreement with non-invasive measurements of adherent cell concentrations calculated from the measured "integrated fluorescent density" minus any background fluorescence. Results show the areal cell concentration (cell number/area) determined from non-destructive direct counts in a pure culture or binary-strain biofilm varied with the biofilm depth. Use of this method to estimate local dynamic plasmid segregational loss and plasmid conjugation transfer kinetics will be reported in a subsequent manuscript.

Energy dispersive X-ray fluorescence determination of cadmium in uranium matrix using Cd Kα line excited by continuum

An energy dispersive X-ray fluorescence method for determination of cadmium (Cd) in uranium (U) matrix using continuum source of excitation was developed. Calibration and sample solutions of cadmium, with and without uranium were prepared by mixing different volumes of standard solutions of cadmium and uranyl nitrate, both prepared in suprapure nitric acid. The concentration of Cd in calibration solutions and samples was in the range of 6 to 90 µg/mL whereas the concentration of Cd with respect to U ranged from 90 to 700 µg/g of U. From the calibration solutions and samples containing uranium, the major matrix uranium was selectively extracted using 30% tri-n-butyl phosphate in dodecane. Fixed volumes (1.5 mL) of aqueous phases thus obtained were taken directly in specially designed in-house fabricated leak proof Perspex sample cells for the energy dispersive X-ray fluorescence measurements and calibration plots were made by plotting Cd Kα intensity against respective Cd concentration. For the calibration solutions not having uranium, the energy dispersive X-ray fluorescence spectra were measured without any extraction and Cd calibration plots were made accordingly. The results obtained showed a precision of 2% (1 σ) and the results deviated from the expected values by < 4% on average.

Real-Time Partitioning of Octadecyl Rhodamine B into Bead-Supported Lipid Bilayer Membranes Revealing Quantitative Differences in Saturable Binding Sites in DOPC and 1:1:1 DOPC/SM/Cholesterol Membranes

Quantitative analysis of the staining of cell membranes with the cationic amphiphile, octadecyl rhodamine B (R18), is confounded by probe aggregation and changes to the probes' absorption cross section and emission quantum yield. In this paper, flow cytometry, quantum-dot-based fluorescence calibration beads, and FRET were used to examine real-time transfer of R18 from water to two limiting models of the cellular plasma membrane, namely, a single-component disordered membrane, dioleoyl-L-alpha-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol, and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration was raised from 0 to 10 mol % in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that were reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parametrized the propagation of concentration-dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum-albumin-mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions where the critical aggregation concentration (CAC) of the probe is defined as 14 nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to that for DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally, this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.

Quantitative liquid and vapor distribution measurements in evaporating fuel sprays using laser-induced exciplex fluorescence

Fully quantitative two-dimensional measurements of liquid- and vapor-phase fuel distributions (mass per unit volume) from high-pressure direct-injection gasoline injectors are reported for conditions of both slow and rapid vaporization in a heated, high-pressure spray chamber. The measurements employ the coevaporative gasoline-like fluorobenzene (FB)/diethylmethylamine (DEMA)/hexane exciplex tracer/fuel system. In contrast to most previous laser-induced exciplex-fluorescence (LIEF) experiments, the quantitative results here include regions in which liquid and vapor fuel coexist (e.g. near the injector exit). A unique aspect is evaluation of both vapor- and liquid-phase distributions at varying temperature and pressure using only in situ vapor-phase fluorescence calibration measurements at room temperature and atmospheric pressure. This approach draws on recent extensive measurements of the temperature-dependent spectroscopic properties of the FB–DEMA exciplex system, in particular on knowledge of the quantum efficiencies of the vapor-phase and liquid-phase (exciplex) fluorescence. In addition to procedures necessary for quantitative measurements, we discuss corrections for liquid–vapor crosstalk (liquid fluorescence that overlaps the vapor-fluorescence bandpass), the unknown local temperature due to vaporization-induced cooling, and laser-sheet attenuation by scattering and absorption.

Automated multichannel fluorescent whole slide imaging and its application for cytometry

Slide-based image cytometry (SBC) has several advantages over flow cytometry but it is not widely used because of its low throughput, complicated workflow, and high price. Fully automated microscopes became affordable with the advent of whole slide imaging (WSI) and they can be transformed into a cytometer. A MIRAX MIDI automated whole slide imager was used with metal-halide and light emitting diode (LED)-based fluorescent illumination, filter block changer, and a cooled monochrome charge coupled device camera. The MIRAX control software was further developed for fluorescent sample detection, autofocusing, multichannel digitization, and signal correction due to nonuniform illumination. Fluorescent calibration beads were used to verify the linearity of the system. The HistoQuant software package of the MIRAX viewer was used for image segmentation and quantitative analysis. The data was displayed by the histogram, scatter plot, and gallery functions of the same program. Fluorescent samples can be reliably detected, focused, and scanned. The measured integrated fluorescence showed linearity with exposure time and staining intensity. Automated fluorescent WSI with stable LED illumination and high-quality homogeneous fluorescent slides can be used conveniently for SBC.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Identification and quantification of ciprofloxacin in urine through excitation-emission fluorescence and three-way PARAFAC calibration

Due to the second-order advantage, calibration models based on parallel factor analysis (PARAFAC) decomposition of three-way data are becoming important in routine analysis. This work studies the possibility of fitting PARAFAC models with excitation-emission fluorescence data for the determination of ciprofloxacin in human urine. The finally chosen PARAFAC decomposition is built with calibration samples spiked with ciprofloxacin, and with other series of urine samples that were also spiked. One of the series of samples has also another drug because the patient was taking mesalazine. The mesalazine is a fluorescent substance that interferes with the ciprofloxacin. Finally, the procedure is applied to samples of a patient who was being treated with ciprofloxacin. The trueness has been established by the regression “predicted concentration versus added concentration”. The recovery factor is 88.3% for ciprofloxacin in urine, and the mean of the absolute value of the relative errors is 4.2% for 46 test samples. The multivariate sensitivity of the fit calibration model is evaluated by a regression between the loadings of PARAFAC linked to ciprofloxacin versus the true concentration in spiked samples. The multivariate capability of discrimination is near 8 μg L −1 when the probabilities of false non-compliance and false compliance are fixed at 5%.

Simultaneous determination of acetylsalicylic acid, paracetamol and caffeine using solid-phase molecular fluorescence and parallel factor analysis

This paper describes the determination of acetylsalicylic acid (ASA), paracetamol and caffeine in pharmaceutical formulations using solid-phase molecular fluorescence and second order multivariate calibration. This methodology is applicable even in the presence of unknown interferences and with spectral overlap of the components in the mixture. Parallel factor analysis (PARAFAC) was used for model development, whose effectiveness was demonstrated by analysis of variance (ANOVA). Errors below 10% were obtained for all compounds using an external validation set. Benefits of the new procedures not included in the reference methods such as low cost, no need of sample preparation, simple and fast analysis using fluorescence spectrometer and no generation of waste, make this method very attractive, allowing for the simultaneous determination of compounds with good reproducibility and accuracy.

Rapid determination of thiabendazole in orange extract using excitation-emission matrix fluorescence and second-order calibration based on alternating trilinear decomposition/alternating normalization-weighted error algorithms

A novel approach is proposed for direct quantitative analysis of thiabendazole in the orange extract by using excitation-emission matrix fluorescence coupled with second-order calibration methods based on the alternating trilinear decomposition (ATLD) and the alternating normalization-weighted error (ANWE) algorithms, respectively. The average recoveries of thiabendazole in the orange extract by using ATLD and ANWE with an estimated component number of two were 99.7 ± 3.3% and 103.5 ± 4.1%, respectively. Furthermore, the accuracy of the two algorithms was also evaluated through elliptical joint confidence region (EJCR) tests as well as figures of merit, such as sensitivity (SEN), selectivity (SEL) and limit of detection (LOD). The experimental results demonstrate that both algorithms have been satisfactorily applied to the determination of thiabendazole in orange extract, and the performance of ANWE is slightly better than that of ATLD.

Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.

Simultaneous analysis of riboflavin and aromatic amino acids in beer using fluorescence and multivariate calibration methods

The study demonstrates an application of the front-face fluorescence spectroscopy combined with multivariate regression methods to the analysis of fluorescent beer components. Partial least-squares regressions (PLS1, PLS2, and N-way PLS) were utilized to develop calibration models between synchronous fluorescence spectra and excitation–emission matrices of beers, on one hand, and analytical concentrations of riboflavin and aromatic amino acids, on the other hand. The best results were obtained in the analysis of excitation–emission matrices using the N-way PLS2 method. The respective correlation coefficients, and the values of the root mean-square error of cross-validation (RMSECV), expressed as percentages of the respective mean analytic concentrations, were: 0.963 and 14% for riboflavin, 0.974 and 4% for tryptophan, 0.980 and 4% for tyrosine, and 0.982 and 19% for phenylalanine.

Determination of Sulpiride in Human Urine Using Excitation-Emission Matrix Fluorescence Coupled with Second-order Calibration

This paper proposes a new and effective approach for the quantitative analysis of sulpiride, a significant antipsychotic drug, in human urine samples by the incorporation of excitation-emission matrix (EEM) fluorescence and second-order calibration methodologies based on the alternating fitting residue (AFR) and self-weighted alternating trilinear decomposition (SWATLD) algorithms. With the application of a second-order advantage, the proposed strategy could be utilized for a direct concentration determination of sulpiride with a simple pretreatment step, even in the presence of serious natural fluorescent interferences. The average recoveries of sulpiride in complex urine samples by using AFR and SWATLD with an estimated component number of three were 101.2 +/- 2.1 and 94.4 +/- 0.7%, respectively. Moreover, the accuracy of the two algorithms was also evaluated through elliptical joint confidence region (EJCR) tests as well as the figures of merit, such as sensitivity (SEN), selectivity (SEL) and limit of detection (LOD). The experimental results demonstrated that both algorithms, as promising quantitative alternatives, have been satisfactorily applied to the determination of sulpiride in human urine, but the performance of AFR was slightly better than that of SWATLD.

Quantifying the Surface Coverage of Conjugate Molecules on Functionalized Nanoparticles

Here we present a method to determine the surface coverage or surface density of biological molecules conjugated to nanoparticle surfaces. Electrospray-differential mobility analysis (ES-DMA) is used to determine a coating thickness by measuring the change in the size of gold nanoparticles before and after modification with thiol-derivatized single-stranded DNA. The DNA surface coverage is then obtained from the coating thickness through the use of a simple random coil model. The method requires neither fluorescent tagging nor calibration curves. We believe ES-DMA to be a broadly applicable nanometrology tool for the characterization of biologically conjugated nanoparticles.

Evaluation of a green laser pointer for flow cytometry

Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times.

Sampling efficiency of a single‐cell capillary electrophoresis system

Capillary electrophoresis (CE) combined with a laser-induced fluorescence (LIF) detection scheme is a powerful approach for single-cell analysis. For measurements requiring a high temporal resolution, CE-LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single-cell CE-LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 microJ) and two different pulse focal positions in the xy plane (0-10 microm from the center of the cell). We found the maximum SE at the lowest energy (2 microJ), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single-cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents.

Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells

There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.

Discrepancy in measuring CD4 expression on T‐lymphocytes using fluorescein conjugates in comparison with unimolar CD4‐phycoerythrin conjugates

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROL Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards.

Hypermethylation of RASSF1A in Human and Rhesus Placentas

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.