Reply to Letter to the Editor

Dear Editor, In his letter, the author present results from a previously published article (Parmryd et al., 2003), and comments on its relationship with our recently published manuscript on colocalization (Bolte & Cordelières, 2006). The author used Pearson’s coefficient to quantify colocalization and the coefficients retrieved from specific regions of interest were very low (from 0.060 to 0.452). The use of Pearson’s coefficient in this situation to quantify colocalization is in contrast to the recommendations we made in the review and this is probably the reason why he sent the letter to the editor. The aim of our study was to sensitize the reader about the pitfalls of using exclusively the Pearson’s coefficient to judge colocalization. To this end, we presented related techniques such as Van Steensel’s method or Costes’s randomization (Van Steensel et al., 1996; Costes et al., 2004), which allow conclusions to be drawn when encountering midrange Pearson’s coefficients such as in the paper cited by the author. The presentation of those alternatives could have been placed earlier in the review to prevent such reactions as in the letter. However, this would have totally impaired the review’s clarity. The objective of our response is thus to clarify the findings of our work in the context of this letter. Three specific points were addressed and we would like to give a step by step answer to clarify the matter: In the first part of the letter, the author supposes that we are misunderstanding the meaning of Pearson’s coefficient. He is quoting the sentence: ‘Values other than those close to 1 and especially mid-range coefficients (−0.5 to 0.5) do not allow conclusions to be drawn’ out of its context. The quotation by itself might impair our demonstration. However, the paragraph was referring to the influence of noise on the Pearson’s coefficient: when Poisson noise is added to one of the two channels used in the study, the Pearson’s coefficient is dramatically reduced, tending to zero. In this situation, even though colocalizing pixels exist in the image, the Pearson’s coefficient is unable to reveal their existence. Also, the writer might not have paid attention to Fig. 5 of our paper: we used several illustrations taking into account the most currently encountered situations in cell biology. As stated in the text, partial colocalization, total colocalization while corrupted by noise and exclusion will give rise to a Pearson’s coefficient in the midrange. In these situations Pearson’s coefficient does not permit us to conclude for partial, total but noise corrupted colocalization, or exclusion. In the second part of his letter, the author states: ‘The suggestion that the Pearson correlation coefficient provides a yes or no answer, with a transition at some unstated value, is wrong’. We have not suggested at any time that Pearson’s coefficient would provide a yes or no answer. We agree with the writer’s statement that associations may not be exclusive, that molecules may not be present in ratiometric numbers and that they may have different spatial distributions. However, we would like to emphasize that Pearson’s coefficient expects the two molecules of interest to be present in ratiometric numbers. Its construction is based on the hypothesis that a linear relationship links their intensities. Trying to apply Pearson’s coefficient in situations not fulfilling this assumption does not, in our opinion, make much sense. In the third part, the author highlights our assumptions on the influence of noise and background on Pearson’s coefficient. He points out that the quality of image acquisition is a major concern on colocalization determination in general and the use of Pearson’s coefficient in particular. We agree with him on this and would like to point out that our review starts with an exhaustive section on acquisition parameters and image quality optimization. We therefore do not understand the author’s concerns when stating ‘this seems to represent a failure to acquire images properly’. Furthermore, our review was not restricted to the use of confocal microscopy in colocalization studies. Wide-field microscopy may also be used, where the background offset is not a parameter that can be set online. Therefore, we do not support the author when he claims that background which impairs Pearson’s coefficient calculation may be minimized by applying an appropriate intensity threshold. We understand the author’s concerns about our propositions, but we emphasize our previous statement that Pearson’s coefficient alone is not recommended any more to perform a proper quantification of colocalization. We respectfully propose that Pearson’s coefficient should be exclusively implied as part of Van Steensel’s or Costes’ method to ensure the statistic relevancy, especially of midrange Pearson’s coefficients.