Ratio Imaging Analyzer (RIA / 莉丫)

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Meet RIA (or as we affectionately call her, "Li Ya / 莉丫").

Ratio Imaging Analyzer (RIA) is a specialized, standalone bio-image analysis software engineered for quantitative ratiometric microscopy. It provides a rigorous, coding-free workflow for processing dual-channel time-lapse data, serving as a powerful alternative to complex ImageJ/Fiji plugins or expensive commercial workstations (e.g., MetaMorph, NIS-Elements).

While originally designed for ratiometric imaging, RIA is fully optimized for Metabolic Imaging and Label-Free Intrinsic Fluorescence studies. It features built-in motion correction (image registration), background subtraction, and standardized data export, making it an essential tool for:

• FRET & Biosensors: Standard CFP/YFP, GFP/RFP, and novel energetic sensors.
• Metabolic Imaging: NAD(P)H/FAD redox ratios and metabolite sensors (ATP, Lactate).
• Label-Free / Structural Biology: Ratiometric Tryptophan (Trp) imaging for protein unfolding/conformational changes.
• Ion Sensing: Calcium (Fura-2, GCaMP), pH (BCECF), and Chloride imaging.

💡 Why use RIA?

• Analysis Unchained: Stop queuing for the lab workstation. RIA is a standalone executable that runs on standard PCs.
• Reproducibility Ready: Science requires verification. RIA allows you to save your entire workspace (ROIs, thresholds, background settings) into a lightweight .ria project file. Send this file to collaborators or reviewers, allowing them to instantly reproduce your analysis and verify your results.
• Math Done Right: Calculating ratios isn't just A / B. Biological images have edges and noise. We implemented a normalized convolution algorithm that handles NaN (Not a Number) values correctly. This means your data doesn't get eroded or corrupted at cell boundaries.
• Zero Coding Needed: We know not everyone loves Python. RIA has a full GUI for background subtraction, thresholding, and dragging-and-dropping ROIs.
• Trust Your Data: We don't hide the numbers. You get the visual stacks, but you also get the raw float32 ratio data and time-series CSVs. You can take these straight to Prism, Origin, or Excel.

🔬 Supported Applications

RIA is algorithmically agnostic to the specific fluorophores, making it compatible with any dual-emission or dual-excitation ratiometric modality:

1. Structural Biology & Protein Dynamics (Tryptophan)

• Intrinsic Tryptophan Fluorescence: Analyze protein conformational changes, unfolding, or binding events by calculating the ratio of emission shifts (e.g., 330nm / 350nm).
• Anisotropy-based Ratios: Process polarized emission channels to study molecular tumbling.

2. Metabolic & Mitochondrial Imaging

• Redox Ratio (Optical Redox Imaging): Quantify metabolic states (Glycolysis vs. OXPHOS) by computing the NADH / FAD autofluorescence ratio.
• Genetically Encoded Metabolite Sensors: Analyze modern biosensors for ATP (e.g., ATeam), Lactate, Pyruvate, or Glucose.

3. FRET & Cell Signaling

• Intermolecular / Intramolecular FRET: Robust processing for standard acceptor/donor emission stacks.
• Kinase Activity Reporters: Track phosphorylation events in real-time (e.g., AKAR, CKAR).

4. Ion & Environmental Sensing

• Calcium Imaging: Ratiometric dyes (Fura-2, Indo-1) and sensors (Ratiometric-Pericam, GCaMP variants).
• pH & Chloride: BCECF, Clomeleon, and other environmental probes.

📁 Project Structure

RatioImagingAnalyzer/
├── data/               # Sample TIFFs so you can try it out immediately
├── paper/              # JOSS submission files
├── src/ria_gui         # The actual code
│   ├── main.py         # Start here
│   ├── gui.py          # The frontend logic
│   ├── processing.py   # The math/algorithm heavy lifting
│   └── components.py   # UI Widgets
├── tests/              # Automated tests to keep bugs away
└── requirements.txt    # Dependencies

🚀 Installation & Editions

RIA is available in two editions to suit different needs: RIA Pro (for advanced features) and RIA Lite (for portability).

💎 Option 1: RIA Pro (PyPI / Source)

Recommended for: Researchers working with .oir, .nd2, .czi files.

The Pro version is the full-featured Python package. It includes comprehensive dependencies (aicsimageio, bioformats) to support reading professional microscopy formats directly.

⚠️ Prerequisite: Java (JDK) To read proprietary formats (like .oir, .nd2) via the Bio-Formats backend, you must have a Java Development Kit (JDK) installed on your system (OpenJDK 11 or later is recommended).

• Exclusive Feature: Direct support for Olympus .oir, Nikon .nd2, and Zeiss .czi files.
• Installation:

  pip install ria-gui

  Once installed, launch it with:

  ria

⚡ Option 2: RIA Lite (Standalone Executable)

Recommended for: Users who want instant access without installing Python.

The Lite version is a re-engineered, lightweight executable optimized for speed and portability. We have significantly reduced the file size and optimized the initialization process for instant startup.

• Best For: Standard TIFF workflows on any Windows PC.
• No Java Required: Since Lite handles standard TIFFs, no external Java environment is needed.
• Key Features: Zero configuration, ultra-fast cold start, minimal memory footprint.
• Download: Check the Releases page to download the latest RIA_Lite_vX.X.exe.

Option 3: Running from Source (Development)

For developers who want to contribute or modify the code:

1. Clone the repository:

   git clone [https://github.com/Epivitae/RatioImagingAnalyzer.git](https://github.com/Epivitae/RatioImagingAnalyzer.git)
   cd RatioImagingAnalyzer

2. Install dependencies: It is recommended to use a virtual environment.

   pip install -r requirements.txt

3. Run the application:

   python src/ria_gui/main.py

📖 Usage Workflow

1. Load Files:
   • Supports both Single-Channel (Intensity) and Multi-Channel (Ratio) Tiff stacks.
   • RIA Pro users can directly drag & drop .oir / .nd2 files (ensure Java is installed).
2. Preprocessing:
   • Motion Correction: Align shaky time-lapse data using the built-in ECC algorithm.
   • Background: Set a global background subtraction (Percentile) or use a custom ROI.
3. Visualization:
   • Switch views between Ratio, Ch1, Ch2, or Aux channels using the toolbar.
4. Analyze:
   • Draw ROIs (Rectangle, Circle, Polygon).
   • Click Plot Curve to see real-time intensity/ratio changes.
5. Save & Export:
   • Save Project: Save your session as a .ria file to allow others to reproduce your work.
   • Export Data: Copy data to clipboard or save processed images as Tiff stacks.

🧪 Automated Testing

RIA v1.8.0 introduces a "Ghost Pilot" automated testing script powered by rich to ensure stability.

To run the visual E2E test demo:

python tests/auto_drive_rich.py

🤝 Contributing

Contributions are welcome! Please check the Issue Tracker or submit a Pull Request.

📄 License

Distributed under the MIT License. See LICENSE for more information.

Citation

If you use RIA in your research, please cite:

Wang, K. (2025). Ratio Imaging Analyzer (RIA): A Lightweight, Standalone Python Tool for Portable Fluorescence Analysis (v1.8.3). Zenodo. https://doi.org/10.5281/zenodo.18107966

Or use the BibTeX entry:

@software{Wang_RIA_2025,
  author = {Wang, Kui},
  title = {{Ratio Imaging Analyzer (RIA): A Lightweight, Standalone Python Tool for Portable Fluorescence Analysis}},
  month = dec,
  year = {2025},
  publisher = {Zenodo},
  version = {v1.8.3},
  doi = {10.5281/zenodo.18091693},
  url = {[https://doi.org/10.5281/zenodo.18107966](https://doi.org/10.5281/zenodo.18091693)}
}