Adolfas K. Gaigalas

Formalization of the MESF Unit of Fluorescence Intensity

This report summarizes the work performed during the past two years at the National Institute of Standards and Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. In addition to the theory underlying the MESF unit, considerations of error analysis are also presented. The details of this work may be found in the three publications of the NIST Journal of Research (www.nist.gov) listed as the references 2–4. The use of the fluorescence intensity unit provides a tool to compare quantitative fluorescence intensity measurements over time and across platforms.

Raman and FTIR Spectroscopies of Fluorescein in Solutions

Raman and Fourier transform-infra red (FT-IR) spectroscopies of fluorescein in aqueous solutions have been investigated in the pH range from 9.1 to 5.4. At pH 9.1 fluorescein is in the dianion form. At pH 5.4, fluorescein is a mixture of monoanion (approximately 85%), dianion and neutral forms (together approximately 15%). The fluorescence quantum yield drops from 0.93 for the dianion form to 0.37 for the monoanion form. The Raman and FT-IR studies focused on the frequency range from 1000 to 1800 cm(-1) which contains the skeletal vibrational modes of the xanthene moiety of fluorescein. At pH 9.1, the spectroscopic feature of fluorescein dianion are consistent with a picture of an electron delocalized among the xanthene moiety and two identical oxygens attached to opposite ends of the xanthene moiety, forming a very symmetric structure. The characteristic of fluorescein dianion is the presence of the phenoxide-like stretch at 1310 cm(-1). At pH 5.4, fluorescein monoanion has lost the symmetric structure characteristic of the dianion. The spectra of the monoanion have distinctive contributions from the phenolic bend at 1184 cm(-1). The assignments of the vibrational bands shown in Raman and FT-IR spectra are given based on both literature and the ab initio calculations at the Hartree-Fock level with HF/6-31 + +G* basis set. Excellent correlation is found between the experimental and calculated spectra.

MOVING SPECTROELECTROCHEMICAL CELL FOR SURFACE RAMAN SPECTROSCOPY

The construction of a moving spectroelectrochemical cell for surface Raman spectroscopy is described. Spectral distortions due to the photodegradation of the surface-bound species are reduced significantly, permitting the use of a higher incident laser power to enhance the Raman signal. The performance of the cell is demonstrated with the surface-enhanced resonance Raman spectra from the protein cytochrome P450cam (CYP101) adsorbed on a silver electrode.

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.

Toward quantitative fluorescence measurements with multicolor flow cytometry

A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF). The objective of this step is to establish a linear relation between the fluorescence signal in a given fluorescence channel of multicolor flow cytometers and the value of ERF. The second step involves a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g., CD4 binding sites). The biological standard is incubated with antibodies labeled with one type of fluorophores for a particular fluorescence channel and serves to translate the ERF scale to an ABC scale. A significant part of the two‐step calibration procedure involves the assignment of ERF values to the different populations of Ultra Rainbow beads. The assignment of ERF values quantifies the relative amount of embedded fluorophore mixture in each bead population. It is crucial to insure that the fluorescence signal in a given range of fluorescence emission wavelengths is related linearly to the assigned values of ERF. The biological standard has to posses a known number of binding sites for a given antibody. In addition, this antibody has to be amenable to labeling with different types of fluorophores associated with various fluorescence channels. The present work suggests that all of the requirements for a successful calibration of a multicolor flow cytometer in terms of ABC values can be fulfilled. The calibration procedure is based on firm scientific foundations so that it is easy to envision future improvements in accuracy and ease of implementation. Published 2007 Wiley‐Liss, Inc.

Quantitating Fluorescence Intensity From Fluorophores: Practical Use of MESF Values

The present work uses fluorescein as the model fluorophore and points out critical steps in the use of MESF (Molecules of Equivalent Soluble Fluorophores) values for quantitative flow cytometric measurements. It has been found that emission spectrum matching between a reference solution and an analyte and normalization by the corresponding extinction coefficient are required for quantifying fluorescence signals using flow cytometers. Because of the use of fluorescein, the pH value of the medium is also critical for accurate MESF assignments. Given that the emission spectrum shapes of microbead suspensions and stained biological cells are not significantly different, the percentage of error due to spectrum mismatch is estimated. We have also found that the emission spectrum of a microbead with a seven-methylene linker between the fluorescein and the bead surface (bead7) provides the best match with the spectra from biological cells. Therefore, bead7 is potentially a better calibration standard for flow cytometers than the existing one that is commercially available and used in the present study.

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.

Diffusionless electron transfer of microperoxidase-11 on gold electrodes

Abstract Microperoxidase-11, MP-11, is made by proteolytic digestion of cytochrome c, cyt. c. It consists of a polypeptide of 11 amino residues attached covalently to the heme. Given that MP-11 has a more exposed heme than the complete protein, it would seem that electron transfer, ET, between immobilized MP-11 and electrodes would be at least as fast as for intact cyt. c. However, while the maximal heterogeneous ET rate for immobilized cyt. c is around 1000 s−1, that reported previously for immobilized MP-11 does not exceed 20 s−1. This work attempts to understand this difference in measured ET rates. The MP-11 was immobilized on gold electrodes using several protocols: (electrode A) the immobilization was done following a previously published carbodiimide based recipe yielding ET rates of the order of 20 s−1; (B) MP-11 was bound to gold electrodes by Lomant’s reagent and gave an ET rate close to 4000 s−1; (C) physisorbed MP-11 on gold electrodes with a self assembled monolayer, SAM, of alkane thiols gave an ET rate approaching 2000 s−1 for the shortest length alkane thiol. Inspection of the immobilization chemistries suggests that the procedure employed in producing electrodes B and C are likely to lead to a monolayer or less of immobilized MP-11 while the procedure employed for electrode A may lead to a film comprised of a multilayer of MP-11. The presence of such a film on electrode A complicates the ET process since the MP-11 in the layer adjacent to the electrode could have fast ET rates while the MP-11 in the outer layers may have significantly slower ET rates. The net result would be an apparent ET rate constant which is much smaller than the value for the first layer. The measurements and calculations are presented in support of such an interpretation.

Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto‐Trol and PBMC–National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto‐Trol is lower and the value on PBMC–NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4+ cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4+ cells from lyophilized PBMC–NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. Published 2012 Wiley Periodicals, Inc.

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.