Quantification in immunofluorescence microscopy a new standard for fluorescein and rhodamine emission measurement

Abstract

In order to obtain microfluorometric standards for fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC), Sephadex G-25 Superfine beads (diameter 10–40 μ) were aminated with aminoethylsulphuric acid and stained with FITC or TRITC. Several parameters of the reaction were assessed. A high degree of linearity was demonstrated between fluorescent staining and: (1) volume of the individual beads; (2) amination time; (3) dye concentration in the staining solutions, and (4) incubation time. The amount of fluorochrome per unit bead volume was determined by measuring the absorption of heavily stained beads. Measurement of the fluorescence emission of the same beads gave a value for the fluorescence per number of fluorochrome molecules. For microfluorometry of biological specimens, beads were prepared with a mean fluorescence of the same order of magnitude as plasma cells stained with anti-immunoglobulin conjugates. These beads showed no significant absorption. It was demonstrated that the ratio fluorescence/number of bound fluorochrome molecules obtained for more heavily stained beads may be extrapolated to lightly stained beads. By comparing beads and plasma cell fluorescence, the latter may be expressed in number of fluorochrome molecules bound to aminoethyl-Sephadex. Bead preparations either stored in glycerol at −20°C or dehydrated were found to be stable over a period of at least several months. The usefulness of this system as a standard in immunofluorescence is discussed with respect to spectral properties of the standard, pH dependency, and quantum efficiency in relation to cells stained with fluorescent antisera.