Computational redesign of a fluorogen activating protein with Rosetta
Fig 3
DiB1, DiB-RM, and DiB-RM-split performance in super-resolution microscopy of living cells.
HeLa cells transiently transfected with vimentin-DiB1 (A, D, E), vimentin-DiB-RM (B, F, G), and transiently co-transfected with vimentin-DiB-RM-splitN1-109 + DiB-RM-splitC110-177-TagBFP constructs (C, H, I) in the presence of 20 nM M739. Imaging conditions: 1.1 kW cm-2 of 488 nm laser light, 30Hz acquisition frequency. (A-C) Super-resolution reconstruction from 10 000 frames, scale bars are 5 μm. (D, F, H) Average projection of 1 000 frames and super-resolution reconstructions from 10 000 frames; scale bars are 0.5 μm. (E, G, I) Normalized intensity profiles between yellow arrows shown on the images (D, F, H); black curve–widefield and red curve–super-resolution. (J-L) Comparison of DiB1, DiB-RM, and DiB-RM-split performance in a localization microscopy setup, average values for 7 cells. (J) Photostability in the localization microscopy setup. (K) The number of detected photons per single-molecule event; vertical lines represent median values. (L) Localization precision per single-molecule event; vertical lines represent median.