Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient
Confocal microscopy is integral to molecular and cellular biology, enabling high-resolution imaging and colocalization studies to elucidate biomolecular interactions in cells. Despite its utility, challenges in handling large datasets, particularly in preprocessing Z-stacks and calculating colocaliz...
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Bio-protocol LLC
2025-04-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5285&type=0 |
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| author | Vikram Aditya Vishakha Tambe Wei Yue |
| author_facet | Vikram Aditya Vishakha Tambe Wei Yue |
| author_sort | Vikram Aditya |
| collection | DOAJ |
| description | Confocal microscopy is integral to molecular and cellular biology, enabling high-resolution imaging and colocalization studies to elucidate biomolecular interactions in cells. Despite its utility, challenges in handling large datasets, particularly in preprocessing Z-stacks and calculating colocalization metrics like the Manders coefficient, limit efficiency and reproducibility. Manually processing large numbers of imaging data for colocalization analysis is prone to observer bias and inefficiencies. This study presents an automated workflow integrating Python-based preprocessing with Fiji ImageJ's BIOP-JACoP plugin to streamline Z-stack refinement and colocalization analysis. We generated an executable Windows application and made it publicly available on GitHub (https://github.com/weiyue99/Yue-Colocalization), allowing even those without Python experience to directly run the Python code required in the current protocol. The workflow systematically removes signal-free Z-slices that sometimes exist at the beginning and/or end of the Z-stacks using auto-thresholding, creates refined substacks, and performs batch analysis to calculate the Manders coefficient. It is designed for high-throughput applications, significantly reducing human error and hands-on time. By ensuring reproducibility and adaptability, this protocol addresses critical gaps in confocal image analysis workflows, facilitating efficient handling of large datasets and offering broad applicability in protein colocalization studies. |
| format | Article |
| id | doaj-art-c5f1c73e242146b7ae2c834dbc075541 |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-c5f1c73e242146b7ae2c834dbc0755412025-08-20T02:26:55ZengBio-protocol LLCBio-Protocol2331-83252025-04-0115710.21769/BioProtoc.5285Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders CoefficientVikram Aditya0Vishakha Tambe1Wei Yue2Department of Pharmaceutical Sciences (VA, VT and WY), University of Oklahoma Health Sciences, Oklahoma City, OK, USADepartment of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USADepartment of Pharmaceutical Sciences (VA, VT and WY), University of Oklahoma Health Sciences, Oklahoma City, OK, USAConfocal microscopy is integral to molecular and cellular biology, enabling high-resolution imaging and colocalization studies to elucidate biomolecular interactions in cells. Despite its utility, challenges in handling large datasets, particularly in preprocessing Z-stacks and calculating colocalization metrics like the Manders coefficient, limit efficiency and reproducibility. Manually processing large numbers of imaging data for colocalization analysis is prone to observer bias and inefficiencies. This study presents an automated workflow integrating Python-based preprocessing with Fiji ImageJ's BIOP-JACoP plugin to streamline Z-stack refinement and colocalization analysis. We generated an executable Windows application and made it publicly available on GitHub (https://github.com/weiyue99/Yue-Colocalization), allowing even those without Python experience to directly run the Python code required in the current protocol. The workflow systematically removes signal-free Z-slices that sometimes exist at the beginning and/or end of the Z-stacks using auto-thresholding, creates refined substacks, and performs batch analysis to calculate the Manders coefficient. It is designed for high-throughput applications, significantly reducing human error and hands-on time. By ensuring reproducibility and adaptability, this protocol addresses critical gaps in confocal image analysis workflows, facilitating efficient handling of large datasets and offering broad applicability in protein colocalization studies.https://bio-protocol.org/en/bpdetail?id=5285&type=0 |
| spellingShingle | Vikram Aditya Vishakha Tambe Wei Yue Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient Bio-Protocol |
| title | Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient |
| title_full | Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient |
| title_fullStr | Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient |
| title_full_unstemmed | Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient |
| title_short | Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient |
| title_sort | development of a novel automated workflow in fiji imagej for batch analysis of confocal imaging data to quantify protein colocalization using manders coefficient |
| url | https://bio-protocol.org/en/bpdetail?id=5285&type=0 |
| work_keys_str_mv | AT vikramaditya developmentofanovelautomatedworkflowinfijiimagejforbatchanalysisofconfocalimagingdatatoquantifyproteincolocalizationusingmanderscoefficient AT vishakhatambe developmentofanovelautomatedworkflowinfijiimagejforbatchanalysisofconfocalimagingdatatoquantifyproteincolocalizationusingmanderscoefficient AT weiyue developmentofanovelautomatedworkflowinfijiimagejforbatchanalysisofconfocalimagingdatatoquantifyproteincolocalizationusingmanderscoefficient |