Systems, methods, and media for calibrating a scanning electron microscope (SEM). The system includes a processor and a memory. The memory stores instructions that, when executed by the processor, configure the system to perform operations. An electron microscope image of a periodic structure is generated by the SEM. A Fourier transform of the electron microscope image is computed to generate a spectrum. Reciprocal lattice vectors are computed based on a known periodicity of the periodic structure. A pixel mask is generated based on the reciprocal lattice vectors and applied to filter the spectrum. A quality metric is generated based on an aggregate magnitude of the filtered spectrum and a magnitude of a zero-frequency component of the filtered spectrum. A pixel scaling parameter, focus parameter, and/or stigmation parameter of the SEM are determined based on the quality metric.