Critical determinants of synaptic functions include subcellular locations, input sources, and specific molecular characteristics. However, there is not yet a reliable and efficient method that can detect synapses. Electron microscopy is a gold-standard method to detect synapses due to its exceedingly high spatial resolution. However, it requires laborious and time-consuming sample preparation and lengthy imaging time with limited labeling methods. Recent advances in various fluorescence microscopy methods have highlighted fluorescence microscopy as a substitute for electron microscopy in reliable synapse detection in a large volume of neural circuits. In particular, array tomography has been verified as a useful tool for neural circuit reconstruction. To further improve array tomography, we developed a novel imaging method, called "structured illumination microscopy on the putative region of interest on ultrathin sections", which enables efficient and accurate detection of synapses-of-interest. Briefly, based on low-magnification conventional fluorescence microscopy images, synapse candidacy was determined. Subsequently, the coordinates of the regions with candidate synapses were imaged using super-resolution structured illumination microscopy. Using this system, synapses from the high-order thalamic nucleus, the posterior medial nucleus in the barrel cortex were rapidly and accurately imaged.