Preparation and microscopic visualization of multicolor luminescent immunophosphors

Abstract

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-

Increasing the luminescence of lanthanide complexes

“…Most luminescent labels based on inorganic elements have large Stokes' shifts as well as narrow emissions, and thus fulfill the first two of the above requirements. Three narrow emitting species of importance as labels are coated semiconductor nanocrystals, generally referred to as quantum dots (10), inorganic phosphor particles (11)(12)(13)(14), and complexes of trivalent lanthanide ions (Ln 31 ) (7,(15)(16)(17)(18). All these labels have one feature in commontheir light-emitting electrons are essentially unaffected by the vibrations of the atomic framework of the surrounding molecular or crystalline structure.…”

“…For flow cytometry and other applications that can employ a focused laser, two-photon absorption of infrared laser light (14,38) is a potentially very useful technique for the excitation of inorganic phosphor particles. The use of these particles is often limited by the background emission resulting from their nonspecific binding (11)(12)(13). However, the elegant coatings (34) developed for quantum dots to inhibit both nonspecific binding and aggregation might be applicable also to phosphor particles.…”

Fast algorithms for the analysis of single and double exponential decay curves with a background term. Application to time‐resolved imaging microscopy

“…To estimate the quality of the fit, two statistical parameters are calculated for each pixel: the residual (equation (15)) and the correlation coefficient (equation (16)) in the minimum of the sum of squares SSQ(min) (defined below in equation (21) [44]). …”

“…Recent innovations in digital imaging microscopy include fluorescence lifetime imaging microscopy (FLIM) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15], delayed luminescence or phosphorescence imaging microscopy [16][17][18] and photobleaching imaging microscopy [19][20][21][22][23][24]. These techniques enable the study of the excited state-photodynamics of fluorophores in the complex environment of a biological specimen with the high resolution and sensitivity of the fluorescence microscope.…”

Lanthanide Nanoparticules as Photoluminescent Reporters

“…Polymerbased lanthanide chelate nanoparticles display improved stability towards pH and quenching of metal ions [92], but they are still moderately susceptible to photobleaching. Thus, inorganic lanthanide-doped nanoparticles should be preferred for luminescence imaging applications [28,94], for which fluorescent nanocrystals such as quantum dots have been already shown to present significant advantages [34] over conventional fluorescent dyes. Inorganic lanthanide-doped nanophosphors possess exceptional stability at high temperature and under harsh conditions; the luminescence intensity of europium(III)-activated yttrium oxysulfide is only slightly affected by temperature change from À20…”