Figure 2: Long time-lapse fluorescence microscopy of the β₂-sliding clamp at the single-cell level utilizing microfluidics.

(a) The microfluidic device used for performing long time-lapse fluorescence microscopy. E. coli cells are immobilized in growth channels perpendicular to a main trench through which growth medium is actively pumped. (inset) A brightfield image and corresponding YPet–β₂ fluorescence image (80 ms laser light exposure) are acquired every 2.5 min for the duration of the time-lapse experiment. Scale bar, 3 μm. (b) YPet–β₂ molecules that are either DNA-bound or freely diffusing are studied using wide-field fluorescence microscopy. (left) Freely diffusing YPet–β₂ molecules in the cytoplasm of a cell. This signal is representative to YPet–β₂ dynamics before and after replication. (middle) A clear focus is observed due to DNA-bound YPet–β₂ molecules. The observation of a single focus, instead of two distinct foci, shortly after initiation results from the overlap of diffraction limited spots. (right) Two distinct foci are visible, indicative of two individual replisomes. Scale bar, 800 nm. (c) Kymograph of a single growth channel during an overnight time-lapse experiment. The cells first grow the growth channel full, and maintain a steady state growth rate as can be observed from the curved shape of the fluorescence signal. The shape of the fluorescence signal is due to the individual cells growing and pushing each other in the direction of the main trench. Clear observable diffuse patterns occur at regular intervals, indicative of no DNA-bound β₂-clamps before initiation or after termination. This repeating pattern is due to the multiple cycles of replication (indicated with repeating white dashed lines). (d) A kymograph of an individual replication cycle indicated in c. The blue lines are the cell boundaries detected from the brightfield images. The illustrations on the right-hand side indicate the different stages of replication that can be observed during the cell cycle.