Quantitating Fluorescence Intensity from Fluorophore: The Definition of MESF Assignment

The quantitation of fluorescence radiance may at first suggest the need to obtain the number of fluorophore that are responsible for the measured fluorescence radiance. This goal is beset by many difficulties since the fluorescence radiance depends on three parameters 1) the probability of absorbing a photon (molar extinction), 2) the number of fluorophores, and 3) the probability of radiative decay of the excited state (quantum yield). If we use the same fluorophore in the reference solution and the analyte then, to a good approximation, the molar extinction drops out from the comparison of fluorescence radiance and we are left with the comparison of fluorescence yield which is defined as the product of fluorophore concentration and the molecular quantum yield. The equality of fluorescence yields from two solutions leads to the notion of equivalent number of fluorophores in the two solutions that is the basis for assignment of MESF (Molecules of Equivalent Soluble Fluorophore) values. We discuss how MESF values are assigned to labeled microbeads and by extension to labeled antibodies, and how these assignments can lead to the estimate of the number of bound antibodies in flow cytometer measurements.     

A user-friendly PC-based data acquisition and analysis system for respirometric investigations

We have developed an easy-to-use computer-based system for recording, displaying, storing and analyzing signals generated by Clark-type oxygen electrodes. A user-friendly interface of Windows-based program BioMed significantly increases the productivity of investigations. It allows to process, control, present and archive the experimental data in real time. A 12-bit analog-to-digital-converter, analog and digital filters, a possibility to zoom the obtained respiratory curves and calculation of the respiration rates by a linear regression method increase the resolution of the estimated oxygen consumption rates. The new system enables to register even small changes, such as 3-5 ngatoms O/min, in respiration rates of biological objects -- enzymes, mitochondria and permeabilized muscle fibers. The system has been developed and is regularly used for the respirometric investigations at the Laboratory of Biochemistry, Institute for Biomedical Research, Kaunas University of Medicine.     

Spectroscopic LSJ notation for atomic levels obtained from relativistic calculations

Today, relativistic calculations are known to provide a very successful means in the study of open-shell atoms and ions. But although accurate atomic data are obtained from these computations, they are traditionally carried out in jj-coupling and, hence, do often not allow for a simple LSJ classification of the atomic levels as needed by experiment. In fact, this lack of providing a proper spectroscopic notation from relativistic structure calculations has recently hampered not only the spectroscopy of medium and heavy elements, but also the interpretation and analysis of inner-shell processes, for which the occurrence of additional vacancies usually leads to a very detailed fine structure.Therefore, in order to facilitate the classification of atomic levels from such computations, here we present a program (within the Ratip environment) which help transform the atomic wave functions from jj-coupled multiconfiguration Dirac-Fock computations into a LS-coupled representation. Beside of a proper LSJ assignment to the atomic levels, the program also supports the full transformation of the wave functions if required for (nonrelativistic) computations.

Program summary

Title of program:LsjCatalogue number: ADTLProgram summary URL:http://cpc.cs.qub.ac.uk/summaries/ADTLProgram obtainable from: CPC Program Library, Queen's University of Belfast, N. IrelandLicensing provisions: NoneComputer for which the new version has been tested: IBM RS 6000, PC Pentium IIIInstallations: University of Kassel (Germany)Operating systems: IBM AIX 4.1.2+, Linux 7.1.+Program language used in the new version: ANSI standard Fortran 90/95Memory required to execute with typical data: Memory requirements depend on the shell structure and the size of the wave function expansion which is used to represent the atomic levelsNo. of bits in a word: All real variables are parametrized by a selected kind parameter and, thus, can easily be adapted to any required precision as supported by the compiler. Presently, the kind parameter is set to double precision (two 32-bit words) in the module rabs_constantNo. of lines in distributed program, including test data, etc.: 225 242No. of bytes in distributed program, including test data, etc.: 3 646 630CPU time required to execute test data: 5 s on a 1 GHz Pentium III processorDistribution format: tar gzip fileKeywords: Atomic, LSJ spectroscopic notation, LS−jj transformation, multiconfiguration Dirac-Fock, recoupling of angular momenta, relativisticNature of the physical problem: The spectroscopic LSJ notation is determined for atomic levels which were calculated previously in the framework of the jj-coupled multiconfiguration Dirac-Fock (MCDF) model. This notation is based on a complete jj−LS transformation of the leading jj-coupled configuration state functions (CSF) in the wave function representation of the selected levels.Restrictions onto the complexity of the problem: The jj→LS transformation of the (jj-coupled) CSF is supported for all shell structures including open s-, p-, d-, and f-shells. For shells with l>3 (i.e. beyond the f-subshells), however, a proper transformation of the antisymmetrized subshell states can be carried out only for the case of one or two equivalent electrons. This restriction also applies for the transformation of the and subshell states which are otherwise supported by the Ratip package [Fritzsche, J. Elec. Spec. Rel. Phen. 114-116 (2001) 1155]. The jj↔LS transformation matrices, which are applied internally by the program, are consistent with the definition of the (reduced) coefficients of fractional parentage [Gaigalas et al., At. Data Nucl. Data Tables 70 (1998) 1; Gaigalas et al., At. Data Nucl. Data Tables 76 (2000) 235] as published previously.Unusual features of the program: The Lsj program is designed as a part of the Ratip package [Fritzsche, Elec. Spec. Rel. Phen. 114-116 (2001) 1155] for the computation of (relativistic) atomic transition and ionization properties. This (new) component therefore supports the transformation of all atomic states which are generated either with Ratip or by means of the Grasp92 code [Parpia et al., Comput. Phys. Comm. 94 (1996) 249]. Moreover, the normalization of the transformed states is tested within the LSJ-coupled basis.     

ris3: A program for relativistic isotope shift calculations

An atomic spectral line is characteristic of the element producing the spectrum. The line also depends on the isotope. The program ris3 (Relativistic Isotope Shift) calculates the electron density at the origin and the normal and specific mass shift parameters. Combining these electronic quantities with available nuclear data, isotope-dependent energy level shifts are determined.     

Measurement of the Fluorescence Quantum Yield Using a Spectrometer With an Integrating Sphere Detector

A method is proposed for measuring the fluorescence quantum yield (QY) using a commercial spectrophotometer with a 150 mm integrating sphere (IS) detector. The IS detector is equipped with an internal cuvette holder so that absorbance measurements can be performed with the cuvette inside the IS. In addition, the spectrophotometer has a cuvette holder outside the IS for performing conventional absorbance measurements. It is shown that the fluorescence quantum yield can be obtained from a combination of absorbance measurements of the buffer and the analyte solution inside and outside the IS detector. Due to the simultaneous detection of incident and fluorescent photons, the absorbance measurements inside the IS need to be adjusted for the wavelength dependence of the photomultiplier detector and the wavelength dependence of the IS magnification factor. An estimate of the fluorescence emission spectrum is needed for proper application of the wavelength-dependent adjustments. Results are presented for fluorescein, quinine sulfate, myoglobin, rhodamine B and erythrosin B. The QY of fluorescein in 0.1 mol/L NaOH was determined as 0.90±0.02 where the uncertainty is equal to the standard deviation of three independent measurements. The method provides a convenient and rapid estimate of the fluorescence quantum yield. Refinements of the measurement model and the characteristics of the IS detector can in principle yield an accurate value of the absolute fluorescence quantum yield.     

The Development of Fluorescence Intensity Standards

The use of fluorescence as an analytical technique has been growing over the last 20 years. A major factor in inhibiting more rapid growth has been the inability to make comparable fluorescence intensity measurements across laboratories. NIST recognizes the need to develop and provide primary fluorescence intensity standard (FIS) reference materials to the scientific and technical communities involved in these assays. The critical component of the effort will be the cooperation between the Federal laboratories, the manufacturers, and the technical personnel who will use the fluorescence intensity standards. We realize that the development and use of FIS will have to overcome many difficulties. However, as we outline in this article, the development of FIS is feasible.     

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.     

MEASUREMENT OF THE DIELECTRIC CONSTANT OF SLURRIES IN PIPES†

The dielectric constant and the ionic conductivity are measured for a slurry flowing in a conducting pipe. These properties are determined from a measurement of the wavelength, frequency and attenuation of radio waves propagating inside the pipe. A mixing model is used to infer the solids fraction of the slurry.     

Biophotonic Tools in Cell and Tissue Diagnostics

In order to maintain the rapid advance of biophotonics in the U.S. and enhance our competitiveness worldwide, key measurement tools must be in place. As part of a wide-reaching effort to improve the U.S. technology base, the National Institute of Standards and Technology sponsored a workshop titled “Biophotonic tools for cell and tissue diagnostics.” The workshop focused on diagnostic techniques involving the interaction between biological systems and photons. Through invited presentations by industry representatives and panel discussion, near- and far-term measurement needs were evaluated. As a result of this workshop, this document has been prepared on the measurement tools needed for biophotonic cell and tissue diagnostics. This will become a part of the larger measurement road-mapping effort to be presented to the Nation as an assessment of the U.S. Measurement System. The information will be used to highlight measurement needs to the community and to facilitate solutions.     

Modeling of Photochemical Reactions in a Focused Laser Beam,II

A method is described for obtaining the rate constant of the photodegradation process of fluorophores illuminated by a focused laser beam. The explicit kinetic equations, describing the population dynamics of excited singlet and triplet states, are averaged over the illuminated volume to describe the resulting fluorescence signal. The illumination is modulated at frequencies from 1 Hz to 100 Hz. Synchronous detection of the resulting fluorescence yields in-phase and quadrature components. The measurement of the ratio of quadrature to in-phase components at several power levels yields information on the photodegradation rate. Specifically it is shown that the data can be interpreted in a manner which yields the value of the photodegradation rate independently of other parameters entering the model. Experiments are performed with erythrosine B which has a large intersystem crossing rate to the triplet state. Measurements in solutions with different viscosities show that the photodegradation rate depends on the viscosity. This is interpreted as evidence for an intermolecular interaction mechanism. We explore the uncertainty of the estimated photodegradation constant taking into account the uncertainties of the measurements used in the synchronous detection technique.