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### Create ROIs
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The next step is the creation of regions-of-interest in the movies. SIMA provides automatic tools for ROI segmentation (see http://www.losonczylab.org/sima/1.3.2/tutorial.html#segmentation-and-rois). However, these approaches are very data specific and require a lot of fine-tuning for specific data sets. Therefore, the current setup relies on manual creation of ROIs with ImageJ or roibuddy GUI. For ImageJ, simply load the relevant image into ImageJ and use the ROI Manager to create an RoiSet file (e.g. RoiSet.zip). For roibuddy, start the program from the command prompt (`python -m roibuddy`) and then load the dataset created previously. When using roibuddy, the ROIs are saved directly with the dataset. For a detailed tutorial of the roibuddy GUI see http://www.losonczylab.org/sima/1.3.2/roi_buddy.html.
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### 03_SIMA_ExtractTraces
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After defining ROIs, we can proceed with extraction of the corresponding calcium traces. This is demonstrated in the notebook 03_SIMA_ExtractTraces.ipynb (both ImageJ and roibuddy ROIs are supported). The extracted traces are then normalized (DFF or DRR), plotted and saved in HDF5 format.
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## Remaining issues
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