BlotTool: A reproducible command-line workflow for Western blot densitometry

BlotTool v1.0.0 BlotTool is an open-source command-line tool for western blot densitometry. The software implements a deterministic workflow in which quantification is performed using explicit, user-defined regions of interest. This approach reproduces standard FIJI/ImageJ densitometry measurements while enabling scripted execution, version-controlled analysis, and transparent documentation of analytical parameters. In the BlotTool workflow, region-of-interest definitions are provided as structured text files specifying labeled rectangular regions and background regions. By separating region definition from measurement execution, analytical decisions about region placement and background correction can be reviewed, modified, and reproduced independently of the measurement process. For each defined band region, BlotTool computes standard densitometric measurements including mean gray value, integrated density, and background-corrected integrated density. The software also generates annotated overlay images documenting the regions used for quantification. BlotTool v1.0.0 adds bit-depth-aware image inversion for 8-bit and 16-bit integer images, explicit image metadata fields in the output CSV, per-ROI saturation reporting, and additional validation examples for TIFF/JPEG handling and saturation-related quality-control behavior. BlotTool is implemented in Python and provides both a command-line interface and a programmatic API. The internal codebase is organized into modular components responsible for input/output handling, measurement logic, visualization, and command-line execution. Core functionality is validated using a pytest-based test suite. Contents of this release: - Command-line interface for western blot densitometry workflows - FIJI-consistent measurements including mean gray value and integrated density - Background-corrected integrated density using user-defined background regions - Bit-depth-aware image inversion for 8-bit and 16-bit integer images - Output CSV fields documenting source image dtype, inferred bit depth, intensity ceiling, inversion status, ROI coordinates, analytical parameters, and saturation-related quality-control fields - Per-ROI saturation reporting for band and background regions - Automated generation of annotated ROI overlay images documenting region placement - Modular Python implementation (io, measure, visualize, cli) - Example dataset and scripts used for validation against FIJI/ImageJ measurements - Additional validation materials for representative western blot images, TIFF/JPEG handling, and synthetic saturation-control behavior - pytest-based test suite Version 1.0.0 marks the documented command-line interface and CSV input/output schema as stable for the ROI-based densitometry workflow described here. Future releases may add functionality, but breaking changes to the documented CLI arguments, ROI CSV schema, or output column meanings will be reserved for a future major version.