Employment of merocyanine 540 fluorescence to form diaminobenzidine (DAB) oxidation product: a photoconversion method for the visualization of erythrocyte membrane fluidity for light and electron microscopy.

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.