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# Triple Area Intensity

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The Triple Area Intensity plugin in Microscopy Image Browser (MIB) calculates image intensities (mean, min, max, or sum) for three areas defined by materials in a model, such as cytosol, nucleus, and background in cells. Results can be displayed in MIB’s Image View panel, stored in the Annotation layer, or saved as an Excel spreadsheet.

## Overview

This plugin measures intensities across three model materials, enabling comparisons (e.g., cytosol vs. nucleus) with optional background correction. It supports ratio calculations and visualization of connected areas, making it ideal for quantitative analysis in microscopy datasets.

## Usage

The following steps demonstrate how to compare image intensities between cytosol and nucleus, using a background reference.
Access the plugin via: `Menu → Plugins → Intensity Analysis → TripleAreaIntensity`.

### Load Images

- Highlight files in the Directory Contents panel using `Shift` + .
- and select `Combine selected datasets` to load as a 3D/4D stack.

### Create segmentation model

Create Model

- Click Create in the Segmentation panel to start a new model.

Add Nucleus Material

- Click `+` in the Segmentation panel to add a material.
- Name the new material as `Nuclei`

Segment Nuclei

- Use the Brush or Segment-anything model tool in the Segmentation panel to draw nuclei areas.
- Ensure material `Nuclei` is selected in `Add to`.
- Press `Shift`+`A` to add drawn areas to `Nuclei` across all slices.

Add Cytosol Material

- Click `+` to add another material and rename it to `Cytoplasm`.

Segment Cytoplasm

- Use the `Brush` tool to draw cytoplasm areas, ensuring the number of cytoplasm objects matches the number of nuclei.
- Select material `Cytoplasm` in `Add to`.
- Press `Shift`+`A` to add drawn areas to `Cytoplasm` across all slices.

Add Background Material

- Click `+` to add a third material and rename it to `Background`.

Segment Background

- Use the `Brush` tool to draw areas outside cells for background intensity.
- Select material `Background` in `Add to`, ensuring at least one background area per image.
- Press `Shift`+`A` to add drawn areas to `Background` across all slices.

### Configure Plugin

- Start the plugin: `Menu → Plugins → Intensity Analysis → TripleAreaIntensity`.
- Select the color channel and parameter (`Mean`, `Max`, `Min`, `Sum`).
- Set `Calculate → Statistics for material 1` to `Nucleus`.
- Set `Calculate → Statistics for material 2` to `Cytoplasm`.

Info

The plugin calculate image intensity for the selected color channel under Statistics for material 1 and Statistics for material 2

- Check `Background` and select `Background` in the dropdown.

Info

Include `Background` to compensate for uneven illumination within images

- Optionally check Subtract background to adjust intensities.

Subtract background

When Subtract background is in the checked state, the plugin will subtract intensities under background areas from the acquired values

- Check `Calculate ratio: Material 1 / Material 2` for ratio output.

Calculate ratio

When Calculate ratio: Material 1 / Material 2 is in the checked state, the plugin additionally calculates the ratio of intensities after background subtraction.

- Specify a filename for Excel export.

### Run Analysis

Click Continue to calculate intensities.

Results appear in the Image View panel (e.g., Background: 142.3671, Nucleus: 340.2101, Cytosol: 99.7331, with background subtracted).

Annotations are stored in `Menu → Models → Annotations`.

Show connections

If `Show connections` is enabled, connected areas are linked in the `Mask` layer (clear via `Menu → Mask → Clear mask`).

Warning

Ensure the number of cytosol objects matches nuclei for accurate pairing.
Use `Show connections` to verify object relationships visually.

Snapshot of results exported using the Microsoft Excel format

## Additional Options

Additional Thresholding: apply thresholding to a material based on background intensity plus a Threshold shift value.

Export to MATLAB: send results to MATLAB’s main workspace as a structure.

Show connections: visualize paired areas in the `Mask` layer, removable via `Menu → Mask → Clear mask`.

## Credits

Written by Ilya Belevich, University of Helsinki
- Version: 1.3, 25.11.2022
- Email: ilya.belevich@helsinki.fi
- Web: https://researchportal.helsinki.fi/en/persons/ilya-belevich

Includes code by Gunther Struyf.

Back to MIB | User Interface | Plugins | Intensity Analysis