The nanometre spacing between nucleosomes, which are folded together throughout live cell chromatin organisation, regulates DNA binding interaction and navigation of the nuclear landscape. This nanoscale feature of chromatin structure is however ‘invisible’ to diffraction limited light microscopy. Thus, direct observation of the regulatory impact nucleosome proximity has on live cell DNA binding protein dynamics has to date been a difficult task. In recent work we established the phasor approach to histone FLIM-FRET microscopy as a method to spatially map nucleosome spacing in a living cell (J Lou et al., 2019 and L Zhen et al., 2020), and here we couple this super-resolved readout of chromatin structure, with a single particle tracking (SPT) analysis of nuclear protein DNA target search behaviour (AJ McCann and J Lou et al., NAR, 2021). Collectively, this multiplexed microscopy approach provides direct insight into the reciprocal interplay between chromatin nanoscale architecture and nuclear protein DNA binding dynamics.