Stimulation of fluorescence in a small contact region between rat basophil leukemia cells and planar lipid membrane targets by coherent evanescent radiation.

We have included a lipid hapten in lipid monolayers coated on alkylated quartz microscope slides to serve as specific antibody-dependent targets for rat basophil leukemia cells.At room temperature basophils armed with specific monoclonal IgE antibodies adhere tightly to the monolayer only when the monolayers contain lipid hapten and only in the presence of Mg2+ and/or Ca2+.The basophils undergo specific IgE-and haptendependent immunologic degranulation under these conditions.Fluoresceinated IgE antibodies bound to Fc receptors on the basophil and located within -100 nm of the monolayer target fluoresce when excited by laser radiation undergoing total internal reflection within the quartz slide.An optical interference pattern produced by two focused intersecting coherent laser beams having total internal reflection within the quartz slide serves as a control to ensure that all of the emitted fluorescence radiation from the IgE molecules is due t o stimulation by evanescent radiation and not by scattered radiation.The interference pattern is also used for pattern photobleaching experiments to determine the rates of lateral diffusion of IgE molecules bound to the monolayers, in the absence of cells.Specific antibody-dependent adherence of basophils to the monolayer target results in a marked enhancement of IgE fluorescence in the field of the evanescent wave.Microscopic examination of the fluorescence of the fluoresceinated IgE molecules in the region of basophil membrane-monolayer membrane contact reveals an extremely complex pattern, presumably due to cellular filopodia-like structures that are anchored t o the target membrane via IgE molecules bound simultaneously to Fc receptors and lipid haptens.These structures are not observable with conventional epifluorescence microscopy due t o the fluorescence of IgE molecules bound to the basophils in regions other than the interface between the monolayer and basophil membranes.The concentration of IgE molecules in the contact region appears to be high and could give rise to Fc-Fc receptor complex formation or other redistributions of Fc receptors that provide signals for basophil degranulation.