An ImageJ/R pipeline for filopodia dynamics analysis.
For the accompanying manuscript, see:
Urbancic, V., Butler, R., Richier, B., Peter, M., Mason, J., Holt, C. E., Gallop, J. L. 2017. Filopodyan: An Open-Source Pipeline For The Analysis Of Filopodia. http://jcb.rupress.org/content/216/10/3405 DOI: 10.1083/jcb.201705113
The plugin is supplied as a .jar file in (releases).
Please refer to the associated user guide (Filopodyan User Guide.pdf) for information on plugin installation, instructions for use and practical advice. API docs are available (here)
To facilitate downstream analysis, we provide our suite of R scripts for downstream analysis of filopodia properties and fluorescence. Familiarity with R is required. Filepaths, imaging parameters and thresholds for analysis need adjusting to suit specific needs as indicated in code comments.
A masterscript (FilopodyanR_MASTERSCRIPT.R) coordinates the execution of multiple other scripts for data import and initial processing (Module 1), computation of filopodia properties over time (Module 2), and comparison of phenotypes (Module 3). Currently these scripts support comparing filopodia from two conditions (input as separate folders). ColourSchemes.R and GraphingTemplates.R are dependencies for Module 3.
For exploring relationships between parameters using correlation matrices, FilopodyanR Correlations_DataInput.R performs data import and processing (analogous to Masterscript and the associated modules), and FilopodyanR Correlations.R performs matrix computation and visualization. This analysis does not handle different conditions (i.e. single folder input).
Analysis of base fluorescence during filopodia initiation is performed with FilopodyanR BaseF.R. It supports analysis of two channels from the same dataset. Various options are provided for background subtraction (Module 1-2). Based on our experience we generally recommend subtracting boundary background, and normalizing to body fluorescence (also background-subtracted using boundary background).
Cross-correlation analysis to explore the relationship between tip fluorescence intensity and filopodia tip movement is performed by FilopodyanR CCF.R. Code for further analysis of this relationship and how it differs between filopodia within a dataset is provided in three additional modules: CCF_subcluster-analysis.R, CCF_Randomisations.R and MarkovChains.R.