How does a protein search for its target site on DNA? Transcription, replication, and DNA repair all begin with a protein binding to a target site located on a long strand of DNA. Despite continued experimental efforts, the detailed mechanisms by which proteins efficiently locate and bind to their target sites among 106-109 decoy sites are not understood. I propose to apply the CLIC microscopy technique to characterize the essential but unexplored mesoscopic search kinetics on complex DNA substrates. A lack of experimental techniques suitable to studying dynamics on complex DNA substrates has limited our understanding of the effect of the spatial organization of DNA, the complex intra nuclear environment, and the other DNA-bound proteins upon the search process. By systematically building up the complexity of the system – from random coil naked DNA to nuclear extracts from prokaryotic and eukaryotic cells – I will use the CLIC technique to characterize the molecular search on native chromosomal DNA. Further, by enabling individual proteins to be tracked for several seconds, rather than for millisecond intervals, CLIC measurements will characterize the complete search process of individual proteins on DNA. CLIC will enable new spatially and temporally resolved measurements of how proteins move on DNA, whether by hops, jumps, slides, inter-segmental transfer or 3D diffusion. Beyond the protein-DNA search, CLIC measurements can be used to characterize the DNA-DNA search that governs how genes are shuffled between parental copies of DNA and the molecular transport of enzymes and proteins which can dynamically alter the properties of biomaterials.