How to synchronize single-cell timeseries over a population

This guide shows you how to collapse single-cell signal traces into population-averaged time series, aligned to a reference event time.

Reference keys: mean signal, signal response, population average

Prerequisite: You have segmented, tracked, measured, and annotated events for a cell population.

Step 1: Configure the signal plot

  1. Go to the Analyze tab and click Plot signals.

  2. In the Options window, configure the following:

    • Population: The cell population (or pair) to analyze.

    • Class: The column used to segregate cells (e.g., class_event). This determines the “event” vs “no event” grouping.

    • Time of interest: The event time column (e.g., t_event) used to align the traces (t=0).

    • Cmap: (Optional) Select a colormap for the curves.

    • Absolute time: Check this to ignore the event time and synchronize signals using an absolute frame number (set via the slider).

    • Query: (Optional) Enter a pandas query to filter cells (e.g., TRACK_ID > 10).

    • Time calibration: Frame-to-minute conversion factor.

    • Pool projection: Choose how to aggregate the population (mean or median).

    • Min # cells for pool: Minimum number of cells required to calculate a valid data point.

  3. Click Submit.

Step 2: Select the signal

  1. A second window appears (“Select numeric feature”).

  2. Select the measurement you want to plot (e.g., mean_intensity).

  3. Click Set.

Step 3: Interact with the plot

The plot window displays the synchronized signals. You can interact with it using the controls:

  • Grouping: Switch between Well (pooled per well), Position (per position), or Both.

  • Toolbar Buttons:

    • Legend: Toggle legend visibility.

    • Log: Toggle log-scale for Y-axis.

    • CI: Toggle 95% confidence intervals.

    • Cell lines: Toggle display of individual single-cell traces.

    • Export: Save the figure or export tabular data.

  • Class of interest: Filter the displayed curves by class:

    • *: Show all cells.

    • event: Show only cells belonging to the event class (class 0).

    • no event: Show only cells belonging to the non-event class (class 1).

  • Rescale: Manually set a scaling factor for the Y-axis.

  • Single-cell signal alpha: Adjust the transparency of individual cell traces.

  • Select position: Choose which positions/wells to display, either by name (checkboxes) or spatially (clicking on the position map).