Live-cell single RNA imaging reveals bursts of translational frameshifting

Kenneth Lyon; Luis U. Aguilera; Tatsuya Morisaki; Brian Munsky; Timothy J. Stasevich

For visuallization and some quantification, average intensity image trims were created by averaging images of all detected particles of a given species (each centered by their intensity centroid). To compute the average number of ribosomes in a translation site (see Fig. S4 ), the average image trims of translation sites and single mature proteins were fitted to Eq. (2). The average intensities were calculated by integrating the fitted Gaussian. The ratio of the average intensity of translation sites to that of single mature proteins was then calculated to give the average number of ribosomes in a translation site. The average intensity of single RNAs were also calculated using the same procedure ( Fig. 3C , S5, and S6). Fits were susceptible to noise, so we also used an alternative strategy to determine the average intensity of translation sites and RNA that was robust to noise (in all Figs aside from Fig. 3C , S4, S5, and S6). Specifically, the intensity of centered images of RNA or translation sites was calculated to be the average intensity within a centered radial spot of four pixels in diameter minus the average background intensity from a centered ring with an outer diameter of twelve pixels and an inner diameter of eight pixels. Mathematica source code is available upon request.