PIE Outputs¶

Outputs of the PIE experiment can be found in the directory provided by the output_folder path in the setup file. Each output folder includes one or multiple folders corresponding to phases of the experiment, named phase_[phase_num], with all phase-specific data contained within (see here for more information on experimental phases).

Complete time-lapse experiment outputs¶

The final experimental output is created only after running PIE.run_timelapse_analysis(...) or pie run_timelapse_analysis .... It contains setup_file.csv, a complete version (including all parameter values initially left to the default) of the original setup file used in the experiment.

In addition, the main folder contains general experimental outputs (Growth Data, Colony properties across time, and in some experiments Movies), as well as a folder for each experimental phase containing phase-specific outputs.

Growth data¶

growth_rates_combined.csv: contains the growth data and key summaries of colony properties over time for every colony that passes growth rate filtration. Properties are reported for each phase, and are followed by _phase_[phase_num] in the column name.

Colony growth properties reported in **growth_rates_combined.csv**¶
Column	Description
cross_phase_tracking_id	a unique ID that can be used to identify each colony across experimental phases. If there is only one brightfield/phase-contrast experimental phase, this will be identical to time_tracking_id
xy_pos_idx	the index of the imaging position the colony is found in
time_tracking_id	a unique ID that can be used to identify each colony across time points within an experimental phase. If there is only one brightfield/phase-contrast experimental phase, this will be identical to cross_phase_tracking_id
t0	the first timepoint in the window used to estimate growth rate
tfinal	the final timepoint in the window used to estimate growth rate
gr	the growth rate of the colony (the natural log of the number of cell divisions per hour)
lag	the pre-growth lag time of the colony in hours
intercept	the y-intercept of the best-fit line to ln(area) between t0 and tfinal (used for plotting growth rates)
rsq	the R² of the best-fit line to ln(area) between t0 and tfinal
foldX	the fold increase in colony area over the entire course of the phase
mindist	the distance to the closest colony recognized for at least `minimum_growth_time` timepoints
cxm	the mean across timepoints of the pixel x-position of the weighted colony center
cym	the mean across timepoints of the pixel y-position of the weighted colony center

In addition, experiments that include fluorescence data will include fluorescent properties in growth_rates_combined.csv, either summarized or from a single timepoint (depending on the value of the fluor_channel_timepoints parameter); see Colony fluorescence properties across time reported in **colony_properties_combined**.

Finally, experiments with post-phase fluorescence will include a fluorescence classification column for each channel imaged in a phase’s post-phase fluorescence, titled fluor_channel_name``_fluorescence_phase_``phase_num.

Colony properties across time¶

colony_properties_combined.csv: contains the (unfiltered) properties of every colony identified in every timepoint during analysis, and the colony tracking data.

Colony properties across time reported in **colony_properties_combined**¶
Column	Description
cross_phase_tracking_id	a unique ID that can be used to identify each colony across experimental phases. If there is only one brightfield/phase-contrast experimental phase, this will be identical to time_tracking_id
time_tracking_id	a unique ID that can be used to identify each colony across time points within an experimental phase. If there is only one brightfield/phase-contrast experimental phase, this will be identical to cross_phase_tracking_id
main_image_name	the name (without file extension) of the image from which colony segmentation was performed to identify a given colony
phase_num	the number of the phase in which colony segmentation occurred
xy_pos_idx	the index of the imaging position the colony is found in
timepoint	the timepoint in which the colony properties are being measured
time_in_seconds	the time, in seconds, that elapsed between the start of the experiment and the current timepoint. If `timepoint_spacing` is provided, time_in_seconds is calculated based on the number of seconds to the current timepoint, and is identical for a given timepoint all xy_pos_idx values. If `timepoint_spacing` is left blank (i.e. is inferred from file modification times), this will be the time between the last modification time of the main_image_name image and the first image saved in `input_path1`
bb_height	the height of the bounding box containing the colony
bb_width	the width of the bounding box containing the colony
bb_x_left	the left-most pixel position of the colony bounding box
bb_y_top	the top-most pixel position of the colony bounding box
cX	the pixel x-position of the weighted colony center
cY	the pixel y-position of the weighted colony center
major_axis_length	the length of the major axis of an ellipse fitted to the colony outline. Inaccurate for small colonies (area ≤ 5 pixels)
perimeter	the perimeter of the colony
area	the area of the colony
label	the label of the colony in the `colony_mask` image (in the phase_``phase_num``/colony_mask directory) corresponding to the current main_image_name

If fluorescent measurements are made, they will also be listed in colony_properties_combined.csv for every colony. Each fluroescent column will be followed by `fluor_channel_name`; if multiple fluorescent channels are specified, the list of columns below will be reported for each one. All measurements (for both background and colony fluorescence) exclude an approximately 3-pixel-wide strip centered on the colony edge (see the PIE paper for details). Background fluorescence is measured within an area of the colony bounding box (the smallest rectangle containing the identified colony) extended by 5 pixels in all four directions but outside of the colony area itself, or of any other colony within the extended bounding box. Note that measurements made during post-phase fluorescence are recorded as belonging to the phase whose colony segmentation they use.

Colony fluorescence properties across time reported in **colony_properties_combined**¶
Column	Description
back_mean_ppix_flprop	The mean fluorescence level per pixel of the background surrounding the colony
back_med_ppix_flprop	The median fluorescence level per pixel of the background surrounding the colony
back_var_ppix_flprop	The variance in fluorescence level per pixel of the background surrounding the colony
col_mean_ppix_flprop	The mean fluorescence level per pixel of the colony
col_med_ppix_flprop	The median fluorescence level per pixel of the colony
col_upquartile_ppix_flprop	The upper quartile of the fluorescence level per pixel of the colony
col_var_ppix_flprop	The variance in fluorescence level per pixel of the colony

Movies¶

PIE automatically generates a gif-format movie of colony outlines and a colony growth graph for any imaging position index listed in the extended_display_positions parameter using the make_position_movie function (see Creating movies of image analysis results for more details) in the movie folder, with each movie named with the relevant position index.

Phase-specific outputs¶

Phase-specific output folders contain:

growth_rates.csv, which contains the same growth rate data as in growth_rates_combined.csv but only for the phase in question
first_timepoint.txt, which contains the time of the first image (this is used during analysis and in the creation of plot movies)
a folder called positionwise_colony_property_matrices containing csv files for each quantified colony property, tracked across time, for each colony; created only after running the run_timelapse_analysis function. This is phase-specific data from each column of colony_properties_combined.csv (see below) in matrix form, and can be useful for performing custom analyses after running PIE.
phase-specific image analysis outputs (see Running PIE single-image analysis, although without a single_image_colony_centers folder, as this data is saved in the colony properties file); these are created during the analysis of every individual imaging position
filtered_colonies.csv, containing a list of colony IDs for colonies that were filtered out at one or more timepoints over the course of the analysis, with the first timepoint a colony is removed due to a particular filter listed in that filter’s column. This data can be helpful for advanced users exploring the effects of tuning filtration options in the experimental setup file.

Temporary outputs¶

In addition to the files described here, PIE creates a number of temporary output files during runtime that are deleted when the experiment is completed. For example, the track_colonies_single_pos function, which is also run internally during time-lapse experiments, creates a summary file for every position called xy``xy_pos_idx``.parquet in a temporary folder called positionwise_colony_properties.