Fluorescence images were acquired using a Nikon spinning disk confocal system with a 60x oil-immersion objective, equipped with an Andor Ixon EMCCD camera, under the control of the Nikon elements software. Images were processed using the image analysis software ImageJ (http://rsb.info.nih.gov/ij/). Representative confocal micrographs displayed in the figures are maximal intensity projections of the 3D data sets, unless otherwise noted.
Live cell imaging of inclusions expressing the fluorescent protein Clover (Lam et al., 2012 (link)) under the control of the hctA promoter (Grieshaber et al., 2012 (link); Chiarelli et al., 2020 (link)) was achieved using an automated Nikon epifluorescent microscope equipped with an Okolab (http://www.oko-lab.com/live-cell-imaging) temperature controlled stage and an Andor Zyla sCMOS camera (http://www.andor.com). Images were taken every fifteen minutes for 48 hours. Multiple fields of view of multiple wells of a glass bottom 24 well plate were imaged. The fluorescence intensity of each inclusion over time was tracked using the ImageJ plugin Trakmate (Tinevez et al., 2016 (link)) and the results were averaged and plotted using python and matplotlib.
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