Figure 1. Characterization of purified PSmOrange in vitro.
(a) Absorbance and emission spectra of PSmOrange before and after photoswitching with 489 nm LED array. (b) PSmOrange far-red brightness and photostability are plotted against the concentration of the oxidant K3Fe(CN)6. (c) Photoswitching half-times for PSmOrange is plotted against the concentration of K3Fe(CN)6. Half-time at 0.25 mM oxidant is shown by dashed line. (d) Formation of the far-red form over time is plotted for purified PSmOrange protein (in 0.25 mM K3Fe(CN)) and for cytoplasmic PSmOrange inside the indicated mammalian cells. The half-time for the purified protein is indicated by dashed line. (e–f) Photoswitching kinetics for orange (e) and far-red (f) forms of the indicated proteins. (g) PSmOrange initial photoswitching rate is plotted against power of the photoswitching 480/40 nm light. (h, i) Photobleaching half-times for the orange (h) and far-red (i) forms of the indicated fluorescent proteins at different power densities Inset: zoomed in bars for 1,130 mW cm−2 power. The power densities were estimated at the sample. The photobleaching data (h–i) were normalized to the spectral output of the lamp, transmission profile of the filter and dichroic mirror, and absorbance spectra of the proteins. Error bars, s.d. (n = 10 (b, c) and n = 5 (g, h, i)).