We demonstrated the high-spatial-resolution imaging of label-free biological cells using an electron beam excitation-assisted optical (EXA) microscope without irradiation damage by the electron beam. An EXA microscope can be used to observe a specimen with a nanometric light source excited in the $$\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}$$ Si 3 N 4 membrane by an electron beam. The incident electron beam penetrates the $$\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}$$ Si 3 N 4 membrane and damages the specimen. To suppress the irradiation damage of the specimen, we prevented the transmittance of the electron beam by coating the $$\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}$$ Si 3 N 4 membrane with a gold thin film. To obtain an electron beam transmittance through the $$\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}$$ Si 3 N 4 of 0%, a gold film of 15 nm thickness was required. By adding the gold layer, a label-free cellular structure was observed with 135-nm spatial resolution.