Regional IF Analyzer

A GUI tool for analyzing immunofluorescence images with atlas region mapping and automated cell counting.

v8.02.000 Highlights

• Major reliability overhaul of the Paint tool for custom regions:
  • Zones named immediately after drawing now correctly register for counting.
  • Count Cells auto-stops paint mode and converts all strokes (named + auto-default).
  • Full state wipe on every new image load (prevents cross-image leakage).
  • Durable model-coordinate storage so drawings survive zoom/pan.
  • Proper interior filling (binary_fill_holes) + neighborhood zone lookup → accurate counts inside hand-drawn structures.
  • No more duplicate zones in the spreadsheet.
• Paint menu improvements:
  • "Save Paint" moved from File menu to Paint menu and renamed Save Paint Layer.
  • New Load Paint command added to the Paint menu.
  • Save Paint Layer now auto-saves directly into the folder currently open in the left File Browser (smart unique naming, no dialog). The file list refreshes automatically.
  • Load Paint and Import Paint default to the current left File Browser directory.
• Critical stability fix: Closing the "Counting Cells" or "Detecting Cells" progress dialog early (X button) can no longer crash the application. All progress UI calls are now defensive.
• Continuing from v8.01: Modern Blob Detection (default), Smart Suggest (Offline), left File Browser with counted status, automatic dual export (.xlsx + _masked.tif), and portable settings.

v8.01.000 Highlights (previous major release)

• New modern Blob Detection engine (Laplacian of Gaussian) — significantly better results on most immunofluorescence images.
• "Smart Suggest (Offline)" — a fully local, privacy-preserving tool that analyzes your image and recommends better detection parameters (with checkbox selection).
• Live switching between Blob and legacy Watershed detection methods directly in Mask Settings.
• Left-side File Browser pane: Select a folder to see all TIFFs, double-click to load, and see which images have already been counted (✓ indicator).
• Automatic export on Count Cells: {image}.xlsx (with Cell Counts + full Detection Parameters metadata sheet) and {image}_masked.tif (original + red mask overlay).
• Export/Import full detection settings as portable .json files from Mask Settings.
• Improved Autotune buttons that adapt intelligently based on the active detection method.
• Brush Settings dialog now opens automatically when using Add/Remove Cell.

Description

The Regional IF Analyzer is designed to help researchers analyze immunofluorescence images by:

• Overlaying atlas sections onto TIFF images
• Highlighting and naming specific regions of interest
• Detecting and counting cells within defined regions
• Automatic Excel + masked image export on Count Cells (with full parameter metadata)
• Saving annotated images

Installation

Prerequisites

• Python 3.8 or higher
• pip (Python package installer)
• tkinter (usually comes with Python, but may need separate installation on Linux)

On Ubuntu/Debian Linux, you might need to install tkinter separately:

sudo apt-get install python3-tk

Setting Up

1. Clone the repository:

git clone https://github.com/LaingLab/BARCC.git
cd BARCC

2. Install required packages:

pip install -r requirements.txt

Note on Excel exports (recommended): Starting with v8.01 (refined in 8.02), clicking Count Cells automatically saves:

• YourImage.xlsx — Contains two sheets:
  • "Cell Counts" (per region)
  • "Detection Parameters" (complete record of every setting used — excellent for methods/reproducibility)
• YourImage_masked.tif — Original image with the final cell mask (including manual edits) as a semi-transparent red overlay.

For full .xlsx support, install the Excel engines:

pip install openpyxl xlsxwriter

Without them, BARCC falls back to a plain .csv.

Running the Program

1. Navigate to the program directory:

cd Application

2. Run the program:

python Application/barcc.py

Basic Usage

1. Import TIFF Image:

   • Click "File > Import TIFF"
   • Select your TIFF image file

2. Determine Regions

   a. Draw Region of Interest:

   • Click "Paint > Start Paint"
   • Draw a circle around the ROI
   • Once done, click "Paint > Stop Paint"
   • Use "Paint > Save Paint Layer" to auto-save the paint into your current left File Browser folder (or "Load Paint" to reload one).

   b. Import Atlas Section:

   • Click "File > Import Atlas Section"
   • Select your PDF atlas file

3. Align Atlas:

   • Use "Move Atlas" button to position the atlas over your image
   • Use rotation and scaling controls if needed

4. Define Regions:

   • Click on regions to highlight them
   • Name each region when prompted

5. Verify Mask:

   • Click "Mask > Show Mask"
   • Adjust detection with "Mask > Show Mask Settings"
   • Manually add and remove cells under "Mask > Add/Remove Cells"

6. Count Cells:

   • Click "Count Cells" to analyze
   • Save results to Excel when prompted

Common Issues

• If tkinter is missing: Install python3-tk package via your system's package manager
• If images don't load: Ensure your TIFF files are in a compatible format
• For PDF loading issues: Ensure PyMuPDF is properly installed

Support

For issues and feature requests, please open an issue in the GitHub repository.

License