Fig. 3.

Detailed procedure for determining TetraSpeck microsphere positions in three fluorescence channels. Picking of microspheres in red (A) green (B) and blue (C) channels using an algorithm based on the CLEAN algorithm (Appendix A) and 2D Gaussian fit for precise position determination (left images, raw data; right images, processed data overlayed with found microspheres). Symbols in (C) indicate both FluoSpheres and TetraSpecks. (D) Filtered positions (symbols) for microspheres in all three channels. The circles, triangles and squares represent the selected Tetraspeck microspheres respectively in red, green and blue channels. (E) Scatter plot of the TetraSpeck microsphere displacement between the different channels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).