Fig. 3: Validation of single-molecule imaging with M2/MS2 arrays.
From: Live cell imaging of single RNA molecules with fluorogenic Mango II arrays
a Diagram and maximum projection of the M2/MS2-SLx24 array under the control of a dox inducible promoter and labelled with TO1-B (200 nM) and NLS-tdMCP-mCherry. Inset shows zoom of selected cytosolic area depicting colocalising signal. Scale bar = 10 µm and 2 µm inset. b Diagram and maximum projection of the MS2-SLx24 array in the presence of TO1-B (200 nM) and NLS-tdMCP-mCherry. c Diagram and maximum projection of the M2/MS2-SLx24 array in the presence of TO1-B (200 nM). d Diagram and maximum projection of the M2x24 array in the presence of TO1-B (200 nM) and NLS-tdMCP-mCherry. a–d All cells shown are PFA fixed Cos-7 cells, scale bars = 10 µm unless otherwise stated, TO1-B signal (green), mCherry signal (red) and Hoechst 33258 (blue). e Side by side comparison of TO1-B (green) and tdMCP-mCherry (red) labelled trajectories for an M2/MS2-SLx24 single molecule at 2 s and 23 s time points. Trace of the trajectory shown in blue and images taken from (Supplementary Movie 3). Scale bar = 4 µm. f XY axis plot of single molecule trajectory from (e) showing co-movement of fluorescent signal coloured as a function of time, TO1-B (green) and tdMCP-mCherry (red). g Plot of distance between foci localised in the TO1-B and mCherry channels (top) and speed (bottom) against time for the trajectory shown in (e) and (f). Average distance and standard deviation between foci plotted as shaded red line. h Fluorescence intensity distribution of M2/MS2-SLx24 foci from live cell tracking, TO1-B fluorescence (green) mCherry fluorescence (red) MS2-SLx24 + TO1-B background fluorescence (black). n = 17,544, 5881 and 33,370 foci, respectively, from 13 cells. i Signal-to-noise ratio distribution of foci detected in live cell M2/MS2-SLx24 tracking, TO1-B (green) and mCherry (red), n = 17,544 and 5881 respectively from 13 cells.
