Figure 3

Flagellar filaments broken using ultrashort laser pulses do not re-grow. (A,B) Flagellar filament of strain EM800 (ΔfliO ΔflgM PflhD* fliC ^T237C) before (A) and after (B) being broken by an ultrafast laser beam. The cell body is barely visible (highlighted with white dotted line) and the filament shows up large and fuzzy because it is rotating much faster than the image acquisition rate. The white arrow points to the broken filament segment drifting away and out of focus. Scale bars are 2 µm. The full movie is available in Supplementary Materials. (C) Control cells of strain EM800 whose filaments were left intact after 2-color labeling. The green portions of filaments that grew during incubation are clearly distinguishable. (D) Example of a bacterium (EM800) that grew a new flagellum during incubation. The top arrow points to the new filament that grew after the first labeling. The filament is blurry since it was rotating during the exposition. The bottom arrow points to the broken filament (orange) that did not regrow. The continued rotation of the flagellar filament demonstrates that the cell was still alive and potentially able to re-synthesize a new filament. (E) Schematic of the experimental setup. The femtosecond laser is added to the optical axis through a dichroic filter (DF) and focused on the sample with a 100 × 1.3 NA objective. The same objective is used for fluorescence imaging. The sample is illuminated with a broadband light source and a fluorescence cube selects the excitation and emission wavelengths. The bacterial filaments are then visualized using an EMCCD camera.