Intensity Corre lation Analysis
This plugin generates Mander’s coefficients as well as performing Intensity Correlation Analysis as
described by Li et al. To fully understand this analysis you should read:
Li, Qi, Lau, Anthony, Morris, Terence J., Guo, Lin, Fordyce, Christopher B., and Stanley, Elise F. (2004).
A Syntaxin 1, G{alpha}o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal:
Analysis by Quantitative Immunocolocalization. Journal of Neuroscience 24, 4070-4081.
Manders, EEM, Verbeek, FJ., and Aten, JA. (1993). Measurement of co-localisation of objects in dual-
colour confocal images. Journal of Microscopy 169, 375-382.
1. Open your two images (File/Open).
2. You must background subtract each stack:
2a. Select a BG ROI.
2b. Run Plugins/ROI/BG subtract from ROI.
2c. Set threshold if required.
3. Run the ICA plugin (Plugins/Colocalization Analysis/Image
Correlation Analysis)
Channel Combination
Select the combination of channel colour, e.g. a TRITC vs FITC analysis would be Red:Green; a FITC vs
DAPI would be Green: Blue. This determines the colouring given to the ICA plots and the merged image.
Use ROI
Choose a ROI to analyse from the drop down box. Choose either ‘none’ to anlyse the
whole image or “Channel 1:’ to analyse the pixels in both channels within the ROI
selected in channel 1.
Use Thresholds
If this is selected, pixels below the image’s threshold will not be included in the analysis. For
instructions on how to set the threshold, see:
http://rsb.info.nih.gov/ij/docs/menus/image.html#adjust and
http://www.uhnresearch.ca/facilities/wcif/imagej/particle_analysis.htm#threshold
Keep merged ROI
Returns a merged image of the analysed ROI (or full image). The merged colours are determined
by the “Channel combination” selected.
Display Colour Scatter plot
This generates a scatter-plot of red intensities vs green intensities. The colour of the scatter plot pixel
represents the actual colour in the image. This does not tell you the frequency of the