STAR-PROTOCOLS-D-23-00123R1
Description
PY79 sporulation.lif is a raw image file (LAS X program (Leica Microsystems, Wetzlar, Germany)). It contains a time-lapse in DIC ( channel 1) and fluorescence (channel 2) channels of B. subtilis PY79 cells in a nutrient-poor medium in the presence of 0.5 μg/ml uM FM4-64 dye. Frames are 5 min apart in the time-lapse series.Cells initially growing vegetatively in a nutrient-rich medium were placed in a nutrient-poor medium to initiate endospore formation. Dye was added, and cells were placed under an agar pad (to immobilize them). Imaging started 1 hour after the nutrient downshift to minimize total light exposure and dye toxicity.Cells that perform engulfment without signs of defects or damage were cropped and rotated. The cropped images were analyzed using the workflow described in the protocol.Three examples of cropped cells are provided (Cell1, 3, and 7). For each cell, the cropped and rotated cell is shown as a .tif stack (e.g., Cell1_timelapse.tif) and as a montage (e.g., Cell1_montage.txt). Membrane contours in individual tif stacks were analyzed using an active contour detection algorithm (Smith…Vavylonis, Cytoskeleton, 2010, 67:693–705. https://doi.org/10.1002/cm.20481). The resulting “snake” files for the mother cell (e.g., cell 1 MC snakes.txt) or the membranes surrounding the forespore (e.g., cell 1 FS snakes.txt) are also included (see the protocol or Smith et al. for details). To locate the individual cells in PY79 sporulation.lif, open PY79_RoiSet.zip. Recording-2024-01-09-14-34-57.mp4 captures screen recordings as authors open, crop cells, pre-process data, and utilize plugins. PY79_sporulation.ijm is a Fiji macro designed for opening and cropping raw data, preparing it for generating snakes in Filament J.