Three-dimensional observation of the radioactive concanavalin A receptors on the surface of human red blood corpuscles utilizing scanning electron microscope autoradiography.

Abstract

Autoradiography combined with scanning electron microscopy (SEM-ARG) was applied to visualize the Con A receptor sites of human red blood corpuscles. Prepared erythrocytes on a glass slide were coated with emulsion by a dipping technique after evaporation by carbon under clean vacuum condition with a cooling trap of liquid nitrogen. Silver halide crystals in the emulsion were distritubed homogeneously on the surface of the erythrocytes. In these experiments, emulsion degelatinized by centrifugation seemed superior to normal emulsion. The remaining gelatin was removed by distilled water at 45°C after development.Electroconductive staining with tannic acid and osmium tetroxide was effective in obtaining good electron contrast and also in preventing artifacts due to the autoradiographic procedure. The number of developed silver grains on the surface of the red blood cells was proportional to the length of exposure time. After exposure for 8 weeks, the average number of grains was calculated to be 312.46±76.07 (3H-Con A-labeled specimen) or 372.58±106.05 (125I-Con A-labeled specimen) per single red blood corpuscle face. The SEM image of the grains was spherical or filamentous, so it was easy to distinguish the grains from the red blood cell surface. On the specimens labeled with 3H-or 125I-Con A, a large number of developed silver grains were distributed homogeneously on the surface of the red blood cells, but on the cells labeled with the same radioactive Con A containing α-metllyl-d-mannoside, there were almost no grains present on the surface. Therefore, the distribution of the developed silver grains probably represents Con A receptor sites.SEM-ARG seems to be a valuable method for the visualization of receptors on the cell surface, even though several problems still remain.