Figure 4.

Red Broccoli–OBI complexes exhibit increased photostability in cells and in vitro. (a) Red Broccoli–OBI exhibits increased photostability in vitro compared to Red Broccoli–DFHO. To test the in vitro photostability, we examined the fluorescence of Red Broccoli–OBI using continuous illumination in the fluorometer. In these experiments, we used solutions containing 1 μM fluorophore (OBI or DFHO) and 20 μM Red Broccoli. (b) Quantification of in vivo photostability. Red Broccoli–BI loses 50% of its fluorescence signal within ~0.2 s, while the fluorescence of Red Broccoli–OBI maintained >75% of its fluorescence for over 10 s. Error bars indicate s.e.m. for n = 3 cells per condition. (c) OBI exhibits markedly improved photostability in living cells compared to BI. In-cell photostability of Red Broccoli–fluorophores (OBI, and BI 20 μM) was assessed by continuous illumination of cells expressing circular Broccoli at an exposure time of 100 ms for each frame. Photobleaching was performed using light sources of similar intensity: 7.3 mW cm–2 for OBI-incubated cells (TRITC filter cube) and 7.2 mW cm–2 for BI-incubated cells (FITC filter cube). Light intensities were measured using an optical power and energy meter console. Scale bar, 10 μm.