USE OF QUANTITATIVE IMAGE MICROFLUOROMETRY TO FOLLOW FLUORESCENT RICIN INTERNALIZATION IN SINGLE LIVING CELLS

We used microspectrofluorometry and videomicrofluorometry to follow th e binding and internalization of fluorescein-labeled toxic lectin rici n in living Zajdela hepatoma cells. Microspectrofluorometry showed tha t when ricin was specifically labeled on its B-chain with one molecule of fluorescein (AB(F)), its fluorescence spectrum did not alter durin g its binding to the cell surface and subsequent internalization. This enabled us to use image analysis to follow cell internalization of la beled ricin. Accordingly, we measured the appropriate fluorescent cell parameters, comprising total fluorescence intensity, cell surface are a, mean fluorescence intensity and its standard error, and used the me asurements for mono- and biparametric studies of cell fluorescence dis tribution. The results showed that (a) ricin binds two different subpo pulations of Zajdela hepatoma cells, (b) Zajdela hepatoma cells intern alize ricin rapidly and after a relatively stable period of 1-2 ht, in ternalization starts again at 4 hr, and (c) the distribution of intrac ellular fluorescence is heterogeneous and AB(F) accumulates in certain cellular localizations. Our results demonstrate that quantitative mic rofluorometry is an effective and interesting approach for real-time s tudies of macromolecule internalization in living cells.