Fluorochromes With Long-lived Fluorescence As Potential Labels For Pulsed-laser Immunocytofluorometry - Photophysical Characterization of Pyrene Derivatives

An apparatus for laser-induced time-resolved fluorescence measurements, in conjunction with pyrene derivatives endowed with long-lived excited singlet states, was employed for immunocytofluorometric measurements. N-(1-pyrene)maleimide, 1-pyrenesulfonyl chloride and 1-pyreneisothiocyanate were conjugated with immunoglobulin, antimouse-IgG, and the fluorescence decays of both free and conjugated forms were investigated. Bi-exponential decays were obtained in all cases with time constants of the short-lived component in the range 3-4.7 ns and the long-lived one in the range 20-55 ns. Only the spectral distribution of the two components is essentially affected upon conjugation. The persistence of the long-lived component, well above the lifetime of autofluorescence, and of the antibody specificity, as shown by immunodiffusion tests, upon conjugation indicates that this technique could be advantageously adopted in immunocytofluorometry.