Specrax led illuminator

For TLFM, appropriate dilutions of cell cultures were transferred to agarose pads containing the appropriate medium on a microscopy slide and covered with a cover glass attached to a 125-μl Gene Frame (Thermo Fisher Scientific) to hold the cover glass on the microscopy slide. TLFM was performed on a Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a ×60 Plan Apo λ oil objective, a TI-CT-E motorized condenser, and a Nikon DS-Qi2 camera. Green fluorescent protein (GFP) was imaged using a quad-edge dichroic (395/470/550/640 nm) and a fluorescein isothiocyanate (FITC) single emission filter. A SpecraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as the light source, using the 470/24 excitation filter. Temperature was controlled with an cage incubator (Okolab, Ottaviano, Italy).
Images were acquired using NIS-Elements software (Nikon), and the resulting pictures were further handled with the open source software ImageJ. The average cellular fluorescence of cells was determined using the open source software Ilastik (55 (link)), which was trained to robustly identify and segment bacterial cells and exclude debris and out-of-focus cells. Background fluorescence was subtracted using NIS-Elements software.

Two wide-field imaging systems were used to capture images of live cells: a Nikon Ti-E-based system and an Olympus cellSens. Details of our live imaging protocols were described in Karanasios et al., 2013 (link), Karanasios et al., 2016 (link). Briefly, cells were plated onto 60 mm dishes and transiently transfected with the relevant plasmids. After 24 hr, cells were replated onto 22-mm-diameter glass coverslips and used for imaging on the following morning. Throughout live imaging, cells were maintained at 37°C in a full enclosure incubation system. The Nikon Ti-E-based system comprised a Nikon Ti-E microscope, 100x 1.4 N.A. objective (Nikon), SpecraX LED illuminator (Lumencor), 410/504/582/669-Di01 and Di01-R442/514/561 dichroic mirrors (Semrock), Hamamatsu Flash 4.0 sCMOS camera, emission filter wheel (Sutter Instruments) and was controlled using Nikon Elements software. The Olympus cellSens system comprised of Olympus IX83 microscope, 100x 1.49 N.A. objective (Olympus), pE-4000 LED illuminator (CoolLED), Hamamatsu Flash 4.0 sCMOS camera, ZT440-445/488-491-594 dichroic mirror (Chroma), Olympus filter wheels on excitation and emission paths and was controlled using Olympus cellSens software.

Cells for immunofluorescence were grown on glass coverslips and fixed in 3.7% formaldehyde in 200 mM Hepes pH 7.2. Staining for immunofluorescence and digital photography were done as described before (Karanasios et al., 2013 (link)). Live-cell imaging was performed as previously described (Karanasios et al., 2013 (link)) using a Nikon Ti-E-based system. Cells plated onto 60 plastic dishes were transferred onto 22-mm-diameter glass coverslips (BDH) and secured in an imaging chamber with 2 ml of cell medium or starvation medium added as indicated. The assembled imaging chamber was secured onto the microscope stage, and cells were maintained at 37°C using an OKO Labs full enclosure incubation system. The Nikon Ti-E-based system comprised a Nikon Ti-E microscope, 100x 1.4 N.A. objective (Nikon), SpecraX LED illuminator (Lumencor, Beaverton, OR), 410/504/582/669-Di01 and Di01-R442/514/561 dichroic mirrors (Semrock), Hamamatsu Flash 4.0 sCMOS camera, emission filter wheel (Sutter Instruments) and was controlled using Nikon Elements software. Compounds (fluorescent analogue, amino acids, and drugs) were added during imaging by flushing the solution in the imaging chamber with 5 ml of fresh solution containing the indicated additions.