STAR-PROTOCOLS-D-23-00123R1

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.

PY79 sporulation.lif 为原始图像文件，采用LAS X程序（徕卡显微系统公司，德国韦茨拉尔）生成格式。该数据集包含枯草芽孢杆菌PY79菌株在添加0.5 μg/ml FM4-64染料的贫营养培养基中，以差分干涉对比度（Differential Interference Contrast，DIC，通道1）与荧光（通道2）双通道采集的延时成像序列，单帧时间间隔为5分钟。
实验初始阶段，菌株在富营养培养基中进行营养生长，随后被转移至贫营养培养基以诱导内生孢子形成。随后加入染料，并将细胞固定于琼脂垫上实现原位固定。为降低总光照暴露与染料毒性，成像于营养条件切换1小时后启动。
针对完成胞吞作用且无明显缺陷或损伤的细胞，本数据集完成了裁剪与旋转处理。裁剪后的图像将按照本数据集配套方案中的流程开展分析。本数据集提供3例裁剪后的细胞示例（分别为Cell1、Cell3与Cell7）。针对每一例细胞，均附带裁剪旋转后的.tif格式时序栈文件（如Cell1_timelapse.tif）与拼接图文件（如Cell1_montage.txt）。
本数据集采用主动轮廓检测算法（Smith…Vavylonis, Cytoskeleton, 2010, 67:693–705. https://doi.org/10.1002/cm.20481）对单张tif时序栈中的细胞膜轮廓进行分析。同时附带母细胞（如Cell 1 MC snakes.txt）或前芽孢周围膜结构的“蛇形”轮廓文件（如Cell 1 FS snakes.txt），详细说明可参考配套方案或Smith等人的原文。
若需定位PY79 sporulation.lif中的单个细胞，可打开PY79_RoiSet.zip。Recording-2024-01-09-14-34-57.mp4 记录了作者打开、裁剪细胞、预处理数据以及调用插件的屏幕操作全过程。PY79_sporulation.ijm 为一款Fiji宏脚本，用于打开与裁剪原始数据，为Filament J中的蛇形轮廓生成流程做好数据准备。