Fig. 2: Determination of effective labeling efficiency of anti-GFP sdABs in co-cultures of cells expressing RyR2D4365-GFP or U2OS-Nup96-mEGFP.
A MINFLUX 3D image of two adjacent ROIs (green boxes) that were imaged time multiplexed in one imaging run. The left ROI was selected in an U2OS-Nup96-mEGFP cell while the right one was located in a HEK293 cell stably expressing RyR2D4365-GFP. Color indicates z-elevation. B 3D view of MINFLUX localizations in the U2OS-Nup96-mEGFP containing ROI with overlaid templates that identify locations and 3D orientation of NPCs. C Quantitative analysis of NPC data shown in B by overlaying the cumulative histograms of labeled NPC segments (0–16) with predicted curves for various values of pLE and line of best-fit (dashed) at pLE = 60.1 ± 0.2 %. D Across experiments on average an effective labeling efficiency of 51.2% ± 2.3% was obtained (N = 10 MINFLUX data sets from n = 3 biological replicates). The inset shows the average number of site visits per U2OS-Nup96-mEGFP site, which is very close to 1 (1.18 ± 0.09, N = 10 MINFLUX data sets from n = 3 biological replicates). Box plots show the median (center line), the 25th and 75th percentiles (bounds of the box), and the mean (large circular marker). Whiskers extend to the most extreme data points within 1.5x the interquartile range, and all individual data points are displayed as a swarm overlay. Scale Bars A: 2 µm, B: 200 nm. Source data are provided with this paper.
