A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue.This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical andlor structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

No abstract

No abstract

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10(-8) to 5 X 10(-6) mole l-1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560-570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.

The preparation of charge-stabilized suspensions of small phosphor particles (0.14.3 pm) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 pm and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads. o 1992 Wiley-Liss, Inc.Key terms: Delayed fluorescence, phosphorescence, immunophosphors, timeresolved microscopy, multiparameter immunocytochemical staining, far UV excitation fluorescence microscopy INTRODUCTION Fluorescent immunocytochemical assays are increasingly utilized for a variety of applications in pathology, oncology, and genetics, mainly because fluorescent labels are very well suited for simultaneous detection of multiple antigens (1,9,15). A disadvantage of these methods, however, is the fact that their theoretical sensitivity is hardly reached because of confounding nonspecific fluorescence due to autof luorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components of the microscopic system. The contrast between specific fluorescence and autofluorescence can be enhanced by using dyes which are excited a t longer wavelengths and emit in the redhear-infra-red part of the spectrum (16), or better by applying time-resolved microscopy.Autofluorescence generally is a rapidly decaying process with fluorescence lifetimes in the range of 1-100 ns, whereas phosphorescence and delayed luminescence have lifetimes in the range of hundreds of micro-