PIE Inputs

PIE can use any image files (.tiff, .png, .jpg) as inputs.

Imaging conditions

PIE can segment colonies from brightfield or phase-contrast images, and can use fluorescent images to calculate fluorescence levels within segmented colonies.

For brightfield imaging, ideal images for PIE analysis are slightly defocused, with dark outlining surrounding cells that are brighter than the background. For phase-contrast imaging, a wide range of imaging conditions are acceptable. See figures 1-5 in the PIE preprint for example images. Importantly, PIE performs well on low-resolution images.

Setup files

Time-lapse PIE experiments require a single csv-format setup file to run. Some templates for experiment types commonly run in the Siegal lab can be found in sample_PIE_setup_files. See the Setting up time-lapse experiments section for more details on how to create new setup files and the parameters that need to be set.

File naming conventions

Naming of single analyzed image files can be arbitrary.

For time-lapse experiments, file names may contain a label for the imaging position, fluorescence channel, and/or timepoint, in any order or combination; however, the naming convention must be coded into the setup file. Read more about how to encode file-naming conventions into your setup file here.