Gene expression is dynamic and heterogeneous across all cell types. It serves several fundamental functions such as adaptation for changing environment and developing tissue specific phenotype and functionality during development and in adult organism. Control of gene expression mostly occurs at the transcription stage. Two main factors that regulate transcription in high eukaryotes are chromatin organization and transcription factor (TF) protein dynamics. Until recently they were mostly studied by methods that are performed in vitro or/and require ensemble averaging. We have been contributing to a development of new single molecule imaging methods that allow quantifying chromatin organization and TF dynamics in vivo and at single cell/single molecule level. Here we present our methodological developments and apply them to study how chromatin organization and TF dynamics regulate transcription. On the technical side of the study we, first, developed a method of imaging DNA with enhanced resolution in living cells. Super-resolution (SR) point localization based microscopy allows visualizing sub-diffraction organization of cellular structures and their dynamics in vivo. However, until recently almost exclusively proteins were imaged in SR microscopy. We found photoswitching conditions to image with stochastic optical reconstruction microscopy (STORM) DNA directly using the site-specific DNA –binding dye Picogreen (Chapter 2). We achieved a resolution of 50-70 nm which is ~5 fold better than conventional microscopy. Due to the excellent dye preservation we were able to do time lapse SR imaging. This study was a first demonstration of using a site-specific dye for STORM SR imaging in living cells. Photoswitching principle used in SR microscopy can be applied to single molecule (SM) tracking. It provides up to 1000x increase in density of trajectories compared to classic SM tracking. Initially, photoactivatable (PA) fusion proteins were used for high density SR based tracking. However, PA proteins are relatively dim and there is a limited choice of them. We demonstrated a new approach to SR based high density tracking by using organic dyes’ photoswitching to track proteins (Chapter 4). We showed that different dyes can be photoswitched in similar live-cell compatible media on membrane and in different organelles. Together with orthogonal protein labelling schemes this allows us to perform multicolour high density tracking across different compartments. This approach gives flexibility of labelling and increased track length compared to PA-FP SR based tracking. In a separate study, using tracking we developed algorithm which can account for long-living molecules in live cell SR imaging and allows us to correct clustering artefacts (Chapter 5). [...]