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.
Centro Endocrinologia ed Oncologia Sperimentale C.E.O.S.-C.N.R., Napoli, Italy.
A
A
Andreoni
G
G
Bottiroli
A
A
Colasanti
M C
MC
Giangarè
P
P
Riccio
G
G
Roberti
P
P
Vaghi
D000818:Animals; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D007150:Immunohistochemistry / Q000379:methods*; D017879:Isothiocyanates; D007834:Lasers; D008301:Maleimides; D051379:Mice; D011721:Pyrenes; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship; D013441:Sulfinic Acids
D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D017879:Isothiocyanates; D008301:Maleimides; D011721:Pyrenes; D013441:Sulfinic Acids; C043592:maleimide; C044255:sulfonyl chloride