The development of fluorescence intensity standards

Abstract

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.

Application of Principal Component Analysis to Elucidate Experimental and Theoretical Information

“…Taking into account that the emission spectra of organic compounds can provide information about them and about their concentration in mixed liquids, this spectroscopic technique appears as a complementary tool allowing the evaluation of the authenticity of matured alcoholic beverages (Gaigalas et al, 2001;Navas & Jimenez, 1999;Walker, 1987). Fluorescence spectra were collected in the 540-800 nm spectral range, using a spectrofluorometer model USB4000-FL from the Ocean Optics company, coupled to a laser of 514 nm wavelength and cuvette holder by optical fibers.…”

How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

“…Independent of the technique and particular instrumentation applied, a major drawback of fluorescence measurements is the demand to remove instrument-specific effects from the measured raw data that otherwise limit the comparability of fluorescence data across instruments, laboratories, and over time [1][2][3][4]11,12]. Further fluorescenceinherent problems are related to difficulties to accurately measure absolute fluorescence intensities and hence to 337 realize true quantitative measurements [13]. In addition, quantitative fluorometry based on measurements of relative fluorescence intensities is hampered by the sensitivity of the absorption and fluorescence properties of most chromophores to their microenvironment that results in environment-specific absorption and emission spectra as well as molar absorption coefficients, fluorescence quantum yields, and fluorescence lifetimes, respectively.…”

“…Absorptance and quantum yield as well as the spectral shape and position of a chromophore's absorption, excitation and emission spectrum can strongly depend on the dye's microenvironment, e.g. the polarity of the surrounding solvent/matrix molecules, pH as well as the concentration of certain ions in the sample [1][2][3][4][5]13]. Furthermore, specific solvent-solute or matrix-solute interactions can play a role.…”

“…On the other hand, for a set of fluorophores in solution and a set of immobilized fluorophores, e.g. bound to or incorporated into a microparticle, the absorption and fluorescence spectra, molar absorption coefficients, and especially the fluorescence quantum yields can differ significantly [13]. If not properly considered, these differences lead to considerable errors in fluorophore quantification.…”

“…The MESF number does not represent the true amount of dye molecules, but indicates the brightness, i.e. the product of the molar absorption coefficient and the fluorescence quantum yield, of the fluorescent species in solution [13,39]. MESF particles are available with different fluorophores like for instance fluorescein (FITC), PE or allophycocyanin (APC) and are routinely used to align the optics of flow cytometers, to set the detection threshold as well as to check the sensitivity and the linearity of the instruments.…”