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. 2014 Nov 11;9(11):e111851. doi: 10.1371/journal.pone.0111851

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© 2014 Malik et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Pixel intensities from images obtained using identical microscope and camera settings were plotted topographically. A plane slicing through the topographic map at a particular percentage of the total intensity reveals only a small number of high intensity pixel clusters for untransfected cells while several high intensity pixel clusters can be observed for NET23/STING transfected cells. (B) Each high intensity pixel cluster for a particular plane in the cells shown in A is color-coded to visualize how accurately the algorithm distinguishes individual clusters. In setting the algorithm this step was used to optimize the parameters for numbers of pixels between clusters that would result in a merging of the clusters. (C) Several different parameterizations are able to distinguish between untransfected and NET23/STING transfected cells. A range of pixel intensity cutoffs for the plane are tested from 5–20% total pixel intensity (%). Also the number of pixels connecting clusters before merging them (m) and the minimum cluster size in pixels (s) were varied. This confirmed that the algorithm is robust and unbiased as statistically significant differences between the untransfected and NET23/STING transfected cells could be observed for nearly all parameters tested. (D) Histogram showing the shift in the distribution of the number of clusters between untransfected and NET23/STING transfected cells at the final parameters chosen: 15% signal intensities, 20 pixel minimum cluster size, and 3 connecting pixels required for merging. (E) Box and whiskers plot showing the distribution for the data in D and p-value calculated using a Kolmogorov-Smirnov (KS) test. (F) The same parameterization with plotting instead the cluster size medians. A similarly strong difference is observed with p-value calculated using a KS test. (G) Nuclear size was also measured for the cells analyzed and found to not change between the untransfected and NET23/STING transfected cell populations.