Spectroscopic properties of fluorescein in living lymphocytes

Detailed studies have been performed on various spectroscopic properties such as time dependence and excitation wavelength dependence of the fluorescence anisotropy for fluorescein molecules introduced into rat thymus lymphocytes. Experimental results have been found to be well interpreted in terms of the coexistence of two types of dye molecules, i.e., free and bound molecules. The fluorescence spectrum of only the bound molecules has been obtained from the difference in the time-resolved spectra of fluorescence with two polarization directions. The time gate has been set at a sufficiently late time after the excitation, so that the polarization memories of the free molecules are lost. The spectrum thus determined agrees very well with that calculated from the spectral data in the stationary condition. From the above results, we come to the conclusion that the main factors which determine the fluorescence anisotropy inside the cell are the fraction and the anisotropy of the bound dye molecules. Finally, we discuss how these factors are related to biological quantities.