Supplementary Figure 7: Resolution analyses of FLINC-based kinase-activity reporters (KAR).

In each row, the feature can be located in the mean fluorescence (Avg) images, and the enzyme activity detected from the feature is shown in the pcSOFI images, labeled by order of calculation. (A-B) Resolution analyses on lifeact-targeted FLINC-AKAR1, where the PKA activity detected on actin filament under different treatments is quantified by either 2nd or 3rd order pcSOFI. The profile of the filament at the red line was plotted in the graph to the right. The activity profile before and after Fsk/IBMX stimulation of PKA are shown to highlight the sensing of activity during superresolution imaging. After Gaussian fitting of many filament profile lines, the average FWHM the filaments were found to be 179±6 nm (n=7) and 116±6 nm (n=7) for (A) 2nd and (B) 3rd order pcSOFI images, respectively. (C-E) Resolution analyses using filopodia features. In each panel, the mean fluorescence (Avg) and the pcSOFI (2nd or 3rd order) image of the same region of interest are shown, while the profiles of the active feature marked by the red dashed line were plotted in the graph to the right. As demonstrated by a graph and a surface plot to aid the eye at the right end of rows C-E, the joints of two filopodial features can be clearly resolved across 3 pixels, allowing the spatial resolution to be estimated. We find a resolution of 160 nm for the 2nd order pcSOFI with FLINC-AKAR1 (C), a resolution of 107 nm for the 3rd order pcSOFI with FLINC-AKAR1 (D), and a resolution of 160 nm for 2nd order pcSOFI with FLINC-EKAR (E).