Universal Z-Stack Focus Profiler & Slice Evaluator

An automated, modular Fiji/ImageJ macro for batch Z-stack focus profiling and optimal slice range selection across multi-channel 3D microscopy datasets. Supports all major vendor formats (.oif, .nd2, .czi, .tif, .lif, etc.) and handles any number of image channels ($N$ channels) dynamically.

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Requirements & Prerequisites

To run this macro, you will need:

• Fiji (ImageJ) (version 1.54 or later recommended).
• Bio-Formats Plugin: Required to open proprietary microscopy stack formats (.oif, .nd2, .czi, .lif, etc.).

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Features & Modularity

• Multi-Format Support: Select specific proprietary file extensions (.oif, .nd2, .czi, .tif, .lif, .lsm) or process all supported formats in a folder.

• Dynamic $N$-Channel Processing: Automatically detects channel count (1, 2, 3, 4+ channels) and appends individual metrics per channel to the final report.

• Dual Analysis Modes:

  1. Dynamic Threshold Mode: Calculates signal bounds based on a relative threshold above background noise: $$\text{Threshold} = \text{Min} + \text{Cutoff} \times (\text{Max} - \text{Min})$$
  2. Fixed Sliding Window Mode: Identifies the optimal subset of $N$ contiguous slices maximizing cumulative edge focus score.

• Interactive GUI Dialog: Configure parameters (extensions, threshold %, window size, padding, CSV auto-save) at launch without editing code.

  

• Headless Memory Optimization: Auto-clears buffer images to ensure crash-free execution on large batch datasets.

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How to Use

1. Open Fiji/ImageJ.
2. Drag and drop Universal_Z_Stack_Profiler.ijm into Fiji, or add it at the end of the Fiji.app/macros/StartupMacros.fiji.ijm file.
3. Click Run.
4. Configure your analysis parameters in the setup dialog.
5. Select the directory containing your 3D stack images.

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Outputs

Upon execution, the macro generates:

1. Results Table & Auto-Saved CSV: An interactive ImageJ table (and .csv file if enabled) listing peak Z-slices ($Z_{peak}$) and optimal slice counts for every channel of each file.
2. Frequency Distribution Histograms: A standalone bar chart per channel (C1, C2, ..., Cn) visualizing the distribution of optimal slice counts across the entire batch.
3. Global Summary Statistics: The ideal mean number of slices printed to the Log window per channel with total batch counts.

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License

This project is licensed under the MIT License - see the LICENSE file for details.