Incucyte Fiji Tools

This repository contains Fiji (ImageJ) Groovy scripts designed to streamline the processing of image data exported from Incucyte live-cell imaging microscopes.

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Overview

1. generate_stacks.groovy

Generates image stacks from single-channel, single–time-point images exported by the Incucyte software. Works with single- and multi-plate experiments in the same input folder.

• Input: Raw/ directory containing Phase/, Green/, and/or Red/ subfolders.
• Parameters:
  • Time interval (minutes): Set the time interval between frames in minutes.
• Automatic detection: The script automatically detects:
  • The vessel ID (e.g. VID9955) from the exported filenames.
  • The available channels (e.g. Phase, Green, Red).
• Output: One multidimensional image stack per (vessel, well, FOV), named VID9955_B3_1.tif.

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2. generate_stacks_with_drift_correction.groovy

Applies XY drift correction to compensate for small position shifts across time points.

• Input: A multi-channel time-lapse image file previously generated with the generate_stacks.groovy script.
• Parameters:
  • Channel 1–3: Choose the channels to include (Red, Green, Phase, or None)
  • Reference: Select which channel will be used to estimate drift.
• Correction method: Uses the MultiStackReg plugin, which depends on the BIG-EPFL update site.

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Requirements

• Fiji (latest release recommended)
• Install the following update sites:
  • MultiStackReg
  • BIG-EPFL

To enable these:

1. Go to Help > Update...
2. Click Manage update sites
3. Check MultiStackReg and BIG-EPFL
4. Apply changes and restart Fiji.

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Notes & Limitations

• Drift correction works best when the reference channel contains high-contrast, stationary features (e.g. confluent cell layers or stable structures).
• Correction may fail or behave erratically when:
  • Cells are not confluent (too sparse).
  • Signal-to-noise is low.
  • Phase images have poor contrast.

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Example (Drift Correction in Action)

Exporting Data from Incucyte

Correct export settings are essential for proper channel detection and time-lapse reconstruction. A quick summary of the key steps is below:

In the Incucyte Experiment Viewer:

1. Click the “Export images and movies” icon.
2. Under Export Type, select “As Stored”.
3. Choose the image type (only one channel at a time):
   • Phase: only one option (8-bit)
   • Red and Green: choose Uncalibrated (16-bit) instead of calibrated
4. Select the desired scan times, wells, and fields of view to export.
5. The exported filenames must follow the Incucyte standard format, which includes the vessel ID automatically:

   VID9955_Phase_B3_1_00d00h00m.tif
   

   If your Incucyte version requires a manual prefix, set it to the channel name only (Phase, Red, or Green) — the vessel ID will be prepended automatically by the software.
6. Save the files as TIFF (.tif).
7. Organize the exported files in the following folder structure:

• Images/
  • Raw/
    • Phase/
    • Green/
    • Red/

The generate_stacks.groovy script should be run using the Raw folder as input.
All generated image stacks will be saved inside a new generated Stacks folder.