Figure 4. SMAUG analysis for single-molecule motion in a eukaryotic system.

A) Super-resolution reconstruction image of BCR-SiR (magenta) and LAT2-mEos3.2 (green) in a representative B cell pre-stimulation. White: overlapping magenta and green signals. Inset is a higher resolution reconstruction of the 1.5 μm × 1.5 μm white boxed region. Scale bar: 2 μm. B) Super-resolution image of the cell in ‘A’ 12.8 min post-stimulation. White: overlapping magenta and green signals. Inset shows same 1.5 μm × 1.5 μm white boxed region as in ‘A’ at a higher resolution. Scale bar: 2 μm. C) Diffusion coefficient and weight fraction estimates for BCR molecules pre-stimulation and at the end of the measurement. Three distinct clusters are found pre-stimulation, but only two at the end of the measurement. D) Bar graphs showing the mean weight fraction of each identified state as a function of time for the BCR dataset. The bars labeled “Pre” and “End” correspond to the data in ‘C’. All other bars are labeled with the time post-stimulation. Identified mobility states are states whose estimates overlap in diffusion coefficient and weight fraction. A new, slower state (blue) emerges 23 s after antigen stimulation. E) The bar graphs for the weight fractions of LAT2 states over time show that the slowest mobility states (blue and red) increase in weight fraction relative to the faster terms (yellow and purple) suggesting the assembly of the BCR signalosome. F) The bar graphs for the weight fractions of Lyn states over time show that there is no change upon antigen stimulation and a slight overall decrease in mobility of the system starting at ~45 seconds. The full cluster analysis is in SI Fig. S5.