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Colocalization analysis is the most common technique used for quantitative analysis of fluorescence microscopy images. Several metrics have been developed for measuring the colocalization of two probes, including Pearson's correlation coefficient (PCC) and Manders’ correlation coefficient (MCC). However, once measured, the meaning of these measurements can be unclear; interpreting PCC or MCC values requires the ability to evaluate the significance of a particular measurement, or the significance of the difference between two sets of measurements. In previous work, we showed how spatial autocorrelation confounds randomization techniques commonly used for statistical analysis of colocalization data. Here we use computer simulations of biological images to show that the Student's one‐sample t‐test can be used to test the significance of PCC or MCC measurements of colocalization, and the Student's …