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Tips for working with CoLocalizer Pro

Correcting background 

Try Auto mode first. Choose presets depending on the pattern of your images. If your image has low contrast or weak fluorescence, click Low Contrast or Weak Fluorescence radio buttons, respectively. If the image is average contrast and fluorescence, use the default Average preset. There is no need to reset the image if you wish to try an another preset.

 

If the results of your calculations look unexpected and/or do not correspond to the standard values of Pearson's correlation and Manders' overlap (R) coefficients (less than -1.0 and/or more than 1.0 for Pearson's coefficient and out of the range from 0 to 1.0 for Manders' coefficient), switch to Manual mode. In this mode, the easiest way to correct background is to use the Threshold Value. Select a Channel Pair, choose the number, and click Apply. If you see that the background was not completely removed, choose a bigger number and click Apply again.

 

More precise correction can be achieved by using Selected ROI. Select an ROI, representing the area that was supposed to be black but absorbed some fluorescence, and perform background correction. To confirm that correction was done properly (all irrelevant pixels were removed), inspect the Histogram. If you still see some pixels, use the Threshold Value to remove them completely. After you see that no irrelevant pixels remain, you have corrected the background properly and may start calculating colocalization coefficients.

 

When correcting the background using a selected ROI, there is no need to reset the image every time you wish to repeat correction. Simply select a new ROI and perform the background correction again.

 

Remember to use background correction settings consistently when analyzing images in the same study. 

 

Selecting ROI 

If you wish to analyze colocalization in a specific area of the image, choose the ROI tool that will enable the most precise selection of the area you will be examining. Polygon or Lasso serve best for this task.

 

Quickly opening images 

To open an image quickly, drag and drop it onto the CoLocalizer Pro icon - just hold down the Command-Option keys while you drag. You can drop image onto any CoLocalizer Pro icon, whether it is in the Dock, in the Finder's sidebar, or in the Finder itself.

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