Nucleoprotein. lament formation by recombinases is central to homologous recombination. To follow this process, we used fluorescent human Rad51 recombinase to visualize the interactions with double-stranded DNA (dsDNA). Fluorescence imaging revealed that Rad51. lament formation on dsDNA initiates from multiple nucleation points, resulting in Rad51-dsDNA nucleoprotein. laments interspersed with regions of bare DNA. The elastic properties of such heterogeneously coated DNA molecules were assessed by combining force-extension measurements using optical traps with fluorescence microscopy. This combination of single-molecule techniques allows discrimination of segments within an individual DNA molecule and determination of their elastic properties. The nonfluorescent zones of DNA-Rad51 constructs showed the well- known (over) stretching behavior of bare DNA. In contrast, the fluorescent, Rad51-coated zones did not overstretch and Rad51 remained stably bound in a structure that was similar to 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.