How to Measure Cell Interface Intensity in QuPath

This guide walks you through measuring fluorescence intensity at the interface between a target cell (red) and a T cell (green) using QuPath.

Prerequisites

• QuPath installed
• CZI image files ready
• An empty folder for your project

Step 1: Set Up Your QuPath Project

1. Create an empty folder on your computer to serve as the project directory.
2. Open QuPath and create a new project, selecting that empty folder as the project location.
3. Drag your CZI files into the QuPath window — QuPath will automatically split each file into its individual images.

Step 2: Annotate the T Cell

1. Draw a rectangle annotation around the target interaction area: the red (target) cell binding to the green (T cell).

   Important: The rectangle should contain only the T cell (green), not the red cell.

2. Select the magic wand tool from the toolbar.
3. Hold Alt and click to remove any regions within the selection that are not part of the T cell.

Step 3: Generate the T Cell Membrane Ring

1. Go to Object → Annotations → Expand Annotations.
2. Enter -0.8 µm to erode the annotation inward.

This produces a thin ring that traces the full T cell membrane.

Step 4: Define the Cell Interface

1. Draw two lines across the T cell membrane ring to mark the boundaries of the interface region (where the T cell contacts the red cell).
2. Go to Object → Annotations → Split Annotations by Line.

   Note: Do not check the second checkbox in the dialog.

This splits the ring into separate annotation segments.

Step 5: Name the Annotations

Double-click each split annotation in the annotation table on the left panel and rename them:

Repeat this naming convention (incrementing the number) for each cell you analyze.

Step 6: Measure Fluorescence Intensity

1. Go to Analyze → Calculate Features → Add Intensity Features.
2. Configure the settings:
   • Pixel size: 0.071 µm
   • Channel: Green channel
   • Measurement: Mean
   • Apply to: All annotations
3. Click OK to run the analysis.

Step 7: Export the Data

1. Go to Measure → Export Measurements.
2. Select Annotations as the export target.
3. Choose CSV as the file format and save.

Tips

• If you forget where any menu option lives, press Ctrl + L to open the command search bar and type keywords to find it quickly.
• Consistent annotation naming (e.g., cell5-interface, cell6-interface) makes downstream analysis much easier to automate or sort in spreadsheet software.