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High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion
Torsten Kroll1,2, David Schmidt1,3,2, Georg Schwanitz1
1Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
Current Protocols in Cytometry
|July 2, 2016
Summary
High-content analysis (HCA) provides quantitative cell data from microscopy. This study details a protocol and bioinformatics pipeline for high-content screening (HCS) to analyze subcellular features like focal adhesions.
Area of Science:
- Cell Biology
- Bioinformatics
- Microscopy
Background:
- High-content analysis (HCA) transforms microscopy images into quantitative data.
- High-content screening (HCS) combines HCA with high-throughput imaging for chemical or RNAi screening.
- Developing robust HCA and bioinformatics pipelines for HCS is complex.
Purpose of the Study:
- To describe the step-by-step development of an HCA assay protocol.
- To present an HCS bioinformatics analysis pipeline.
- To demonstrate the utility of the developed methods for analyzing subcellular features.
Main Methods:
- Development of a novel HCA assay protocol.
- Creation of a dedicated HCS bioinformatics analysis pipeline.
- Application of the protocol and pipeline to focal adhesion (FA) detection and analysis.
Main Results:
- Successful quantitative analysis of multiple FA features.
- Functional annotation of signaling pathways regulating FA size.
- Demonstration of the protocol's effectiveness on published RNAi screen data.
Conclusions:
- The developed HCA assay protocol and HCS bioinformatics pipeline facilitate quantitative analysis of subcellular features.
- This strategy is valuable for both small-scale microscopy and large HCS experiments.
- Enables researchers to extract biologically meaningful data from microscopy-based studies.
