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SUMMARYWe investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of haptenlabeled DNA probes, membrane antigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

A technique is described to modify a standard fluorescence microscope for time-resolved visualization of delayed luminescing substances with decay times from 50 ps to several milliseconds. The modification consists of synchronized operation of a mechanical shutter, positioned in an aperture plane in the excitation pathway, simultaneously with a ferro-electric liquid crystal (FLC) shutter on the emission side. Operation of the microscope is through a microprocessor interfaced keypad by which all timing parameters can be adjusted for optimal suppression of fast decaying luminescence. Accuracy of the timing was within 1 ps. Prompt fluorescence was suppressed up to lo6 times, as determined for bright prompt fluorescing microspheres.The use of the FLC shutter resulted in a reduction in emission intensity by a factor of 8 (due to the use of polarizers, the lower transmission of the FLC devices, and IR blocking filters). No significant image degradation due to shutter operations was observed. The modified microscope was successfully used for the visualization of delayed luminescing immunolabels, such as inorganic phosphor particles and lanthanide chelates, as well as naturally phosphorescing materials.

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-