One milliliter of a stationary culture of E. coli were pelleted by centrifugation and resuspended in PBS. NBD-DDA and the membrane dye SynaptoRed C2 (Sigma-Aldrich, S6689) were added to a final concentration of 10 μM. The suspension was applied to an object slide and covered with a no. 1.5H cover slip (Marienfeld). Imaging was done on a confocal laser scanning microscope Leica SP8 equipped with a white light laser (Superk Extreme EXW-9 NIM, NKT Photonics, Denmark) using a 100x oil immersion objective with a numerical aperture of 1.4. The fluorescence of NBD-DDA was excited at 470 nm (laser power of 34%) and recorded in the spectral window of 500–600 nm. The membrane stain SynaptoRed C2 used for colocalization studies was excited at 570 nm (laser power 24.6%) and detected in the spectral window of 650–800 nm. Both channels were sequentially excited to avoid spectral bleed through. The pixel size of the recorded images was 41.4 nm.
A z-stack with 10 images and a step size of 0.26 μm was recorded. The images were deconvoluted with the software Huygens Essential (Version 17.04, Scientific Volume Imaging B.V., The Netherlands) with default settings and a maximum intensity projection of the deconvoluted z-stacks was created. The intensity histograms of the images were adjusted to improve the visibility of the fluorescence signal.
A z-stack with 10 images and a step size of 0.26 μm was recorded. The images were deconvoluted with the software Huygens Essential (Version 17.04, Scientific Volume Imaging B.V., The Netherlands) with default settings and a maximum intensity projection of the deconvoluted z-stacks was created. The intensity histograms of the images were adjusted to improve the visibility of the fluorescence signal.