We measured the fluorescence lifetime by a new method (Itoh et al., 1997a) from cross-correlation signals at an optical point on the fluorescence image of human melanoma cultivated cells stained by the DNA probe acridine orange (AO). As the lifetime values from the phase-delay of the cross-correlation signals were almost the same as the modulation by a framing camera, the method was applicable to taking ultra fast time-resolved fluorescent images. The obtained shorter lifetime (1.1-2.3 nsec)-resolved image of the red fluorescent region was about the same as that from the fluorescence lifetime obtained in 1.98 nsec by the laser flash photolysis method. lt was considered that the shorter lifetime region in the cytoplasm particle was in the cytoplasm that included single-stranded RNA of living melanoma cells.
We established a simple method to evaluate the ratio of protein loading in cultured dorsal root ganglion (DRG) neurons. Fluorescein isothiocyanate (FITC)-conjugated dextran was used as a tracer of protein loading. Antibodies and FITC-dextran were concomitantly introduced into chick DRG neurons using a trituration method (by which macromolecules can be mechanically introduced into cell populations using pipettes). A correlation between the antibody loading and the fluorescence of FITC-dextran was determined quantitatively. Complete retention of the loaded antibody for 3 hours after trituration was confirmed immunocytochemically in approximately 71% of the FITC-dextran-positive cells. The ratio of the loaded antibody correlated quantitatively with the fluorescence intensity of FITC-dextran in a single cell. Trituration loading of proteins can be indirectly visualized and quantitatively evaluated by the fluorescence of FITC-dextran concomitantly loaded with proteins into neurons in primary culture. We use this method to monitor protein loading in living cultured DRG neurons.