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An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images
Published on: August 31, 2012
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A comparative study of 2 computer-assisted methods of quantifying brightfield microscopy images
1The University of Edinburgh/MRC Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh, UK.
Applied Immunohistochemistry & Molecular Morphology : AIMM
|January 25, 2013
Summary
This study compares Adobe Photoshop and ImageJ for immunohistochemistry image analysis. For DAB staining, both software packages perform similarly, but Photoshop excels with complex stains like Picrosirius Red (PSR).
Area of Science:
- Digital pathology and image analysis in biomedical research.
- Quantitative assessment of immunohistochemical staining.
- Comparative analysis of scientific image processing software.
Background:
- Immunohistochemistry is vital for antigen detection, relying on image analysis software.
- Existing software can be complex, costly, and require advanced computer skills.
- Objective comparison of accessible software for quantitative staining analysis is needed.
Purpose of the Study:
- To compare the efficacy of Adobe Photoshop CS6 and ImageJ for quantifying positive staining.
- To evaluate software performance with two common staining methods: 3,3'-diaminobenzidine (DAB) and Picrosirius Red (PSR).
- To determine which software is superior for analyzing images with varying levels of positive staining.
Main Methods:
- Quantitative analysis of DAB-stained mouse spleen sections (B cells) and PSR-stained mouse kidney allografts.
- Image analysis using Adobe Photoshop CS6 and ImageJ to measure percentage positive area.
- Statistical comparison using Student's t-test, correlation coefficients, and Bland-Altman plots.
Main Results:
- No significant difference was found between Photoshop and ImageJ for analyzing DAB-stained tissues (P=0.243, r=0.985).
- Photoshop demonstrated superior performance for PSR-stained tissues, better analyzing low positive pixel areas and maintaining differentiation.
- Bland-Altman analysis showed poor agreement (r=0.804) between the methods for images with higher positive staining percentages.
Conclusions:
- For 3,3'-diaminobenzidine (DAB) stained tissue image analysis, Adobe Photoshop and ImageJ offer comparable results.
- Adobe Photoshop is superior for analyzing complex color stains like Picrosirius Red (PSR), especially in differentiating low positive staining areas.
- Software choice impacts quantitative accuracy, particularly for nuanced staining patterns and control samples in immunohistochemistry.
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