The intricate physical interaction of transcription factors with specific target genes in the genome has long been regarded as a fundamental mechanism of cell-type specific gene expression. However, due to insufficient spatiotemporal resolution of microscopy techniques, direct visualization of protein dynamics within the nuclei of living cells had not been achieved for decades. Resolving existing limitations, recent advances in imaging techniques enabled the direct observation of protein dynamics, even at a single-molecule level. In addition, the new imaging techniques accomplished capturing higher-order chromatin structures that influence gene expression regulation along with transcription factor dynamics. This review discusses the recent applications of microscopy techniques to investigate the dynamics of nuclear proteins in living cells and their achievements.