Microenvironment of endosomal aqueous phase investigated by the mobility of microparticles using fluorescence correlation spectroscopy

Temporal observation of the dynamic behavior of molecules in cells gives information about the physiological environment at the region of interest. Here we report the direct measurement of the mobility of rhodamine-labeled microparticles (14 and 35 nm in diameter) ingested in endosomes of cultured bovine aortic endothelial cells using fluorescence correlation spectroscopy (FCS). The fluctuation of fluorescent signals from microparticles were measured by FCS. Obtained autocorrelation functions (FAFs) were analyzed by the 2-D multicomponent model according to an evaluation procedure we newly developed. It was found that microparticles moved freely in endosomes with average diffusion coefficients of 4.3 x 10(-8) and 2.7 x 10(-8) cm(2) s(-1) for 14 and 35 nm, which were 45% slower than in water. This result implies that the endosomal aqueous phase is homogeneous with the viscosity about 2.2 times of water. Our study also proposes the new use of FCS for investigation of the internal space of organelles. (C) 2001 Academic Press.