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. 2015 Jul 1;5:10270. doi: 10.1038/srep10270

Figure 2. YPet is the optimal yellow FRET acceptor for cyan donors.

Figure 2

a. Cyan FPs have differential photostabilities under nominal excitation. COS-7 cells were transfected with cytosolic CyPet and continuously imaged for 20 frames (100 ms per acquisition) with 442 nm excitation (125 μW illumination). All scale bars display 5 μm. b. Normalized intensity traces for ROI around individual cells expressing different cyan donors. Notice that CyPet exhibits two photostable states, which is problematic for imaging cellular dynamics on the timescale of seconds to minutes. Error bars report standard deviation with N = 20. c. COS-7 cells were transfected with Tq2-YPet and the acceptor was selectively photobleached by collimated 515 nm laser illumination (1,500 ms pulse at 72 W/cm2; 15 cycles). d. The average intensity for ROIs around individual cells over the time course of the acquisition. FRET efficiency is calculated as the change in donor intensity before and after the acceptor is photobleached, resulting in a FRET efficiency of 0.73 ± 0.02. e. Summary of characteristic FRET efficiencies for various cyan/yellow pairs showing selection of YPet as the best yellow acceptor. All error bars are standard deviation with N = 30. All bars are statistically significant (p < 0.05), using Two-way ANOVA followed by Tukey HSD comparison of means, except where noted. f. COS-7 cells expressing various cyan/yellow pairs were treated with 100 μg/mL cycloheximide (CHX) for 5 hrs in order to block synthesis of new protein. Linked molecules are assumed to be expressed at equal ratios; however, because of the differential maturation rates of the FPs this may not the case. Indeed, prior to treatment with CHX Tq2-YPet has a [Y] to [C] ratio of 1.32 and TFP-YPet is 1.58. Following treatment with CHX for 5 hours, the [Y] to [C] ratio is 1 to 1 for all constructs. All error bars are standard deviation with N = 10. Ratios before and after CHX treatment not connected by the same letter are statistically significant (p < 0.05), using Two-way ANOVA followed by Tukey HSD comparison of means. (Significance for E[DA]/[A] and E[DA]/[D] are not shown for clarity.)