Single-molecule imaging, a super-resolution live imaging method, has enabled direct tracking of nanoscale movements of individual proteins in living cells. However, its application has been limited to dissociated cell cultures due to technical constraints, which hindered the investigation of individual protein behavior within intact tissues. To overcome this limitation, we aimed to develop a method for single-molecule imaging within tissue specimens. We introduced a novel chemical tag technology named De-QODE, consisting of a small-molecular QODE probe and DeQODE protein tag. The QODE probe, initially non-fluorescent, becomes highly fluorescent upon reversible binding to the DeQODE tag. These unique properties allow for fluorescent labeling of proteins of interest with remarkably low-background fluorescence even within tissue samples. By harnessing De-QODE-based single-molecule imaging, we successfully achieved high-density tracking of synaptic molecules in neurons within acutely isolated brain slices. This groundbreaking approach provides unprecedented insights into the dynamic behavior of proteins within the intricate tissue environment, significantly advancing our understanding of cellular processes in their natural context.