Fluorescence imaging of DNA replication. (A) Schematic representation of the E. coli DNA-replication machinery. Coordinated unwinding of parental double-stranded DNA and synthesis of two daughter duplexes is catalyzed by a large multiprotein complex, the replisome, built up from 12 different proteins and held together by a large number of weak and strong protein–protein and protein–DNA interactions. (B–F) Quantitative characterization of the number of polymerases per replisome in living E. coli using single-molecule slim-field microscopy. (B) Laser light is focused on the back aperture of the microscope objective, generating an intense Gaussian field at the sample just large enough to image a single E. coli cell. (C and D) Overlay of bright-field images of cells (gray) and 90-ms frame-averaged fluorescence images (yellow) of fluorescently labeled polymerases (ε-YPet). The blue arrows point at replisomes with three polymerases and the red arrow indicates a replisome with six polymerases. (E) Raw (blue) and filtered (red) intensity for a putative single (left panel) and double (middle panel) replisome spot were compared with the intensity of a single surface-immobilized YPet in vitro (right panel). Combined with the Fourier spectral analysis to find the brightness of a single YPet (F), these data show that the in vivo steps were integer multiples of the intensity of a single YPet molecule and replisomes contain a mean of three polymerases. (B–F are adapted with permission from Reyes-Lamothe et al. (2010).) (G) Two-color fluorescence imaging of the concentration-dependent exchange of ssDNA binding proteins on ssDNA. A microfluidic flow cell with ssDNA curtains was alternatingly injected with RPA-mCherry (magenta) and E. coli ssDNA binding protein (SSB)-EGFP (green). The exchange is evident by the change in color of the fluorescence and length of the ssDNA. Arrows placed above the kymograph indicate the time points of the injections. (G is adapted from Gibb et al. [2014].)