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FIA

DOI

FIA (Fluorescence Image Aligner) is a robust motion correction plugin designed to solve the full spectrum of motion artifacts in biological time-lapse microscopy.

Developed by Dr. Kui Wang at the Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Chinese Academy of Sciences, FIA bridges the gap between high-precision rigid alignment and non-rigid deformation correction. It is specialized for Calcium Imaging (G/R-CaMP), Voltage Imaging, and long-term live cell monitoring.

Author: Kui Wang, CEBSIT, CAS. For questions please use the GitHub Issues or tag @Epivitae on image.sc.

Key Features

  • Dual-Engine Core:
    • OpenCV ECC: Best for high-precision, sub-pixel rigid alignment (Rotation/Translation).
    • Dense Flow (New Standard): State-of-the-art local deformation correction using Farnebäck optical flow. Handles internal tissue warping and soft tissue deformation better than rigid methods.
  • Scientific Integrity (Intensity Preservation):
    • FIA guarantees that your ΔF/F analysis remains valid.
    • Navigation: Motion vectors are calculated using noise-reduced (CLAHE), contrast-enhanced temporary frames.
    • Transport: These vectors are applied to your original raw data using cubic interpolation. No artificial contrast is written to your final image.
  • Smart “Super Reference”:
    • In Dense Flow mode, FIA creates a reference anchor by averaging N frames. This eliminates the “floating anchor” problem caused by shot noise in single-frame references.

Installation & Update

FIA is distributed via the Biosensor Tools Fiji Update Site.

  1. Open Fiji / ImageJ.
  2. Navigate to Help › Update…
  3. Click Manage update sites.
  4. Check Biosensor Tools from the list.
  5. Click Apply and Close, then restart Fiji.

If the site is missing from the list, you can add it manually:
Name: Biosensor Tools
URL: https://sites.imagej.net/Biosensor-Tools/

Usage Guide

1. Mode Selection

FIA offers three operational modes tailored to different biological samples:

Mode Type Best Application
Global Rigid / Affine General Drift Correction. Corrects XY translation and rotation. Recommended for behaving animal tracking or whole-brain imaging.
Dense Flow Non-Rigid (Optical Flow) 95% of Biological Samples. Uses Super Reference + CLAHE. Best for noisy fluorescence, brain slices, and in vivo imaging where local deformation occurs.
Elastic Non-Rigid (Raw) High-SNR Data. Uses raw optical flow without preprocessing. Best for binary masks, artificial beads, or clean data where contrast enhancement is unnecessary.

2. Parameter Tuning

The Smart UI adapts based on your selected mode.

For Dense Flow Mode:

  • Flow WinSize: The “field of view” for local alignment.
    • Small (5): Captures fine jitter.
    • Large (15+): Captures global tissue waves.
  • Ref Depth: Number of frames averaged to create the “Super Reference” (Default: 5). Higher values reduce noise in the reference anchor.
  • Poly N: Smoothing factor. 5 provides sharper motion borders, while 7 offers smoother flow fields but may blur distinct motion boundaries.

For Global Mode:

  • Max Iterations: The loop limit for Rigid/Affine calculation.
  • Precision: Convergence threshold (epsilon).

Algorithms

FIA is built upon established computer vision algorithms to ensure reliability:

  1. Non-Rigid Registration: Based on Farnebäck’s polynomial expansion (2003) combined with CLAHE (Contrast Limited Adaptive Histogram Equalization) for robustness against uneven illumination.
  2. Rigid Alignment: Utilizes Parametric Image Alignment using Enhanced Correlation Coefficient (ECC) Maximization (Evangelidis & Psarakis, 2008).
  3. Legacy Stabilization: Includes a fallback engine based on the classic Lucas-Kanade optical flow.

Citation & DOI

Wang, K. (2026). FIA: Fluorescence Image Aligner - Robust Motion Correction for ImageJ/Fiji. Zenodo. https://doi.org/10.5281/zenodo.18206931

Developed by Kui Wang © 2026. Part of the Biosensor Tools suite.