Fluorescence Properties of Fluorescein Isothioyanate (FITC) Immobilized on Microbeads
Adolfas Gaigalas, Lili Wang, A. Schwartz
We report measurements of the fluorescence properties of fluorescein isothiocyanate (FITC) immobilized on microbeads with spacers of three different lengths. Both the wavelength of maximum emission and the wavelength of maximum absorption shift towards longer wavelengths as the length of the spacer decreases. The formalism developed by McRea was used to correlate the spectral shifts in terms of the effective optical and dielectric properties at the solvent and microbead interface. Polarization measurements suggest that the motion of the FITC is more constrained for shorter spacers. The fluorescence lifetimes of immobilized FITC have minimal dependence on the spacer length. The constancy of the life time implies a constant quantum yield which suggests that the change in spacer length does not modify the internal conversion relaxation mechanism. Comparison of the excitation and emission spectra of FITC labeled microbeads to that of FITC labeled whole blood cells suggests that the spacer with 12 carbons gives the best match of spectral properties. The results point to a right path for improving quantitation in fluorescence based clinical assays.