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 excit ed singlet states, was employed for immunocytofluorometric measurement s. N-(1-pyrene)maleimide, 1-pyrenesulfonyl chloride and 1-pyreneisothi ocyanate were conjugated with immunoglobulin, antimouse-IgG, and the f luorescence 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 o f the long-lived component, well above the lifetime of autofluorescenc e, and of the antibody specificity, as shown by immunodiffusion tests, upon conjugation indicates that this technique could be advantageousl y adopted in immunocytofluorometry.