Cellular function relies on the dynamic transport and interaction of biomolecules within a highly crowded intracellular environment. Pair correlation microscopy offers a minimally invasive and highly sensitive approach to quantitatively map the diffusive routes of fluorescent proteins in living cells with single-molecule resolution. At the core of this method is the pair correlation function (pCF), which compares temporal fluorescence intensity fluctuations between two spatially distinct points in a scanned image series. This enables direct measurement of how molecular transport is influenced by intracellular architecture across multiple spatial scales. Despite its potential, the broader application of pCF has been limited by the complexity of data analysis. To overcome this, we recently developed a streamlined, reproducible protocol for pCF acquisition and analysis1. Here, we apply this protocol to investigate how transcription factors (TFs) navigate the nuclear landscape in the context of genome organisation. Using live mouse embryonic stem cells, we track the dynamics of Sox2, Oct4, and Nanog following acute degradation of the structural proteins CTCF and Rad21, which are essential for topologically associating domain (TAD) formation. Our results reveal that TF diffusion is highly sensitive to TAD disruption, showing altered behaviour at both short and long spatial ranges. Moreover, we demonstrate that loss of TAD integrity impacts TF-chromatin interactions, offering mechanistic insight into how these structural domains modulate the frequency of transcriptional bursts. This work positions pCF as a pivotal tool for connecting nuclear architecture with molecular dynamics and provides new insights into how genome structure modulates protein mobility and gene regulation in real-time.