Ti ct e motorized condenser

Fluorescence microscopy was used to screen 200 mutants for nucleoid reporter activity. All fluorescence microscopy and time-lapse fluorescence microscopy experiments were performed with a temperature controlled (Okolab Ottaviano, Italy) Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a TI-CT-E motorized condenser, a YFP filter (Ex 500/24 nm, DM 520 nm, Em 542/27 nm), a DAPI filter (Ex 377/50 nm, DM 409 nm, Em 447/60), and a CoolSnap HQ2 FireWire CCD-camera. For imaging, cells were grown to mid-log phase and placed between LB agar pads and a cover glass, essentially as described previously [20] (link), and incubated at 37°C. Where appropriate, DAPI was added in the LB agar pad at a final concentration of 1 μg/mL, chloramphenicol at a final concentration of 2 μg/mL, nalidixic acid at a final concentration of 150 μg/mL and rifampicin at a final concentration of 100 μg/mL. Mitomycin C was added to the liquid culture 30 min prior to imaging at a final concentration of 1 μg/mL.
Images were acquired using NIS-Elements (Nikon) and resulting pictures were further handled with open source software ImageJ (downloaded from http://rsbweb.nih.gov/ij/).

For TLFM, cell suspensions were diluted appropriately, transferred to agarose pads (containing the appropriate medium), placed on a microscopy slide, and mounted with a cover glass. A Gene Frame (Thermo Scientific) was used to hold the cover glass on the microscopy slide. TLFM was performed with a temperature controlled (Okolab, Ottaviano, Italy; 37 °C) Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a 60× objective, a TI-CT-E motorized condenser, a YFP filter (Ex 500/24, DM 520, Em 542/27), a CFP filter (Ex 438/24, DM 458, Em 483/32), a GFP filter (Ex 472/30, Dm 495, Em 520/35), an mCherry filter (Ex 562/40, Dm 593, Em 641/75), a DAPI filter (Ex 377/50, DM 409, Em 447/60), and a CoolSnap HQ2 FireWire CCD-camera. Images were acquired at user-chosen time intervals using NIS-elements software (Nikon). During the acquisition of TLFM recordings, care was taken to prevent potential photobleaching of fluorescent molecules (i.e., the photochemical alteration of a fluorophore molecule by, for example, the prolonged exposure light of excitation wavelengths, such that it permanently is unable to fluoresce) by minimizing excitation light intensity and enlarging time intervals in between acquisition of fluorescent images. The resulting images were further handled with open source software ImageJ.