3D-SIM imaging of microtubules using tyrosination sensor. (A) Z-stack of 3D-SIM reconstructed images of N-terminal TagRFP-T tagged to binder A1aY1 (red tyrosination sensor, shown in green here) stably expressed in interphase U2OS cells and labeled with SiR-tubulin (red; scale bar = 2 µm). The boxed areas are magnified below each panel. (B) Colocalization colormap contains spatial distribution of calculated normalized mean deviation product (nMDP) values (ranging from −1 to 1). The distribution is based on a color scale in which negative nMDP values are represented by cold colors (segregation); values above 0 are represented by hot colors (colocalization). Representative frequency scatter plot for colocalization and pseudo-colormap of correlations between pairs of corresponding pixels in TagRFP-T_A1aY1 and SiR-tubulin images, thereby offering quantitative visualization of colocalization. (C) Line scans of representative single microtubules, from the position shown in the insets with a yellow line, demonstrating the relative resolutions of 3D-SIM and widefield images. (D) Quantification of FWHM measurements of n = 50 microtubules for 3D-SIM and widefield (WF) images for both SiR-tubulin and red tyrosination sensor (TagRFP-T A1aY1). A.U., arbitrary units.