Fig. 2

Screening of blue mRuby3 variants, in vitro characterization of two best performing mutants. (a) Intracellular brightness and photostability half-times of 67 variants expressed in HEK cells using pHybrid-/mScarlet expression under screening conditions. Imaging conditions for intracellular brightness of BFP variants: 403 nm LED excitation, 456 nm emission; for half-life measurement: 403 nm LED excitation, 456 nm emission (Supplementary Table S2). (b) Blue-to-red fluorescence ratio in live HEK cells expressing 7 best performing blue mRuby3 variants using pHybrid-/mScarlet (n = 73, 71, 48, 89, 80, 72, 55 cells from one transfection; Kruskal–Wallis ANOVA p-value = 8.4e-8; Supplementary Table S3; imaging conditions for BFPs: 403 nm LED excitation, 456 nm emission, 0.91 mW/mm2 power). Narrow part of notch, median; top and bottom of the notch, 95% confidence interval for the median; top and bottom horizontal lines, 25% and 75% percentiles of the data; whiskers extend 1.5 × IQR from the 25th and 75th percentiles; horizontal line, mean; dots, outliers. Subplot shows intracellular brightness (y’y) and photostability half-times (x’x) resulted from (b, c). Electra1 is shown in light blue, Electra2 in dark blue throughout. (c) Time dependent intracellular fluorescence as part of the screening process in HEK cells using pHybrid-/mScarlet as expression vector (n = 10, 10, 10, 10, 11, 11, 11 cells from one transfection; imaging conditions for BFPs: 403 nm LED excitation, 456 nm emission; 4.25 mW/mm2 power). (d, e) In vitro excitation and emission spectra of Electra1 and Electra2, respectively. Excitation was measured in the range 250 nm-500 nm and emission from 420 to 700 nm for both purified proteins; Ex peak at 402 nm for Electra1; Ex peak at 403 nm for Electra2; Em peak at 456 nm for both proteins.