Fluorometry of turbid and absorbant samples and the membrane fluidity of intact erythrocytes

In employing intrinsic or extrinsic fluorophores in the study of whole cells, or other strongly absorbant and/or scattering samples, the measured fluorescence intensity and polarization is seriously affected by absorption and scattering within the sample cuvet. These artifacts are analyzed and simple protocols are provided for overcoming them. An expression relating attenuation of the observed emission anisotropy to sample turbidity is derived. The validity of the method is confirmed by experiments in which the emission anisotropies and fluorescence yields of membrane probes in intact erythrocytes was measured with precision. It is also shown that the rotational mobility of the membrane probe 1-phenyl-3-(2-naphthyl)-2-pyrazoline is the same for intact erythrocytes and ghosts. These protocols are particularly useful in measuring the intrinsic fluorescence yield ratio for excimeric and monomeric emission of pyrene-containing membrane probes. This provides a method for determining the local lateral mobility of excimeric probes in intact erythrocytes