Automated

In this human cytoplasm-nucleus translocation assay, learn how to load a previously calculated illumination correction function for two separate channels, measure protein content in the nucleus and cytoplasm, and calculate the ratio as a measure of translocation. This is a clumpy cell type, so studying the settings in primary object identification may be helpful for users interested in the more advanced options that module offers. More about these images can be found at the BBBC.

This protocol first extracts the cell nuclei from a given fluorescence channel (full labeling), and grows a contour from each nucleus to extract the cell edge in another fluorescence channel (membrane-labeling).

The Macro processes a composite picture in ImageJ/Fiji and outputs a color-balanced merged RGB image.

To calculate the white balance, a rectangle at coordinates (x=100, y=100) and of size (w=100 pixels, h=100 pixels) is used. These values can be changed to make sure that a background region is taken for the calculation in the line: makeRectangle(100,100,100,100). The user could be prompted to draw the region by removing the signs // in the line: // waitForUser("Please draw a region in the background");