Figure 3

Photostability of StayGold, EGFP, mClover3 and mNeonGreen fused to H2B in living and fixed HeLa cells in HBSS under continuous WF (unattenuated arc lamp) illumination. Irradiance: 2.2 W/cm². (a) left, Photobleaching curves of the four green-emitting FPs in live-cell samples. middle, Photobleaching curves of the four green-emitting FPs in fixed-cell samples. In each photobleaching experiment, five cells were observed (indicated in the first images, insets). Scale bars, 50 µm. right, Photobleaching curves (green lines: live cells; gray lines: fixed cells) were simply normalized; FI_(t)/FI₍₀₎ was plotted against time. Data points are shown as means ± SD (n = 5 cells). (b) Comparison of the photostability of the four green-emitting FPs in living cells. Photobleaching curves are calculated based on the irradiance and FP molecular brightness (Table 1), plotted as intensity versus normalized total exposure time with an initial emission rate of 1000 photons/s/molecule. Data points are shown as means (n = 5 cells). (c) Comparison of the photostability of the four green-emitting FPs in fixed cells. As the molecular brightness of FPs in their fixed state has not been determined, simply normalized photobleaching curves shown in (a, right) were collected. Data points are shown as means (n = 5 cells). (a, c) Cells were treated with 4% PFA for 30 min. See Fig. 2.