Accumulating evidence suggests that molecular dynamics at nanometer scale is crucial for synaptic functions. Single-molecule fluorescence imaging is a super-resolution live imaging method that enables direct tracking of movement of individual molecules. However, conventional single-molecule imaging has been applicable only to dissociated cells on coverslips due to technical limitations, preventing the analysis of events that occur only in the intact brain tissue. In this study, we set out to develop a method for single-molecule imaging within brain slices and the brain in vivo. We developed and employed a novel chemical tag technology named De-QODE. This technology consists of a small-molecular QODE probe and DeQODE protein tag. Non-fluorescent QODE becomes highly fluorescent upon reversible binding to DeQODE. These properties realize fluorescent labeling of proteins of interest with extremely low-background fluorescence even within tissue samples. Furthermore, De-QODE-based labeling is repeatable after photobleaching. Owing to De-QODE-based single-molecule imaging, we succeeded in high-density tracking of synaptic molecules in pyramidal neurons deep within acute cortical slices.