ABSTRACTWe present the development of a custom‐built structured illumination microscope (SIM) featuring a specially designed transmission diffraction grating. Employing the analog microfilming method, we fabricated transmission diffraction gratings suited to the specific requirements of our system. This robust and cost‐effective method allows for the fabrication of diffraction gratings with customized constants, ensuring excellent transmission in both the visible and near‐infrared spectra. Additionally, to assess the performance of our system, we measured the resolution in both epifluorescent and super‐resolution imaging modalities by applying two independent methods: the conventional resolution measurement using fluorescent beads and the knife‐edge technique applied on the MoS2 monolayer flakes. Both methods confirmed enhancement in the resolution of SIM over the epifluorescent imaging modality. Furthermore, we have successfully demonstrated the capabilities of our microscope by imaging fluorescently labeled astrocytes, specifically targeting the vimentin filament protein within these cells. The super‐resolution images reveal fine structures of the vimentin cytoskeleton that remain unresolved in the epifluorescent image.