StayGold photostability under different illumination modes

Abstract

StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.

Figure 1

Classification of fluorescence microscopy. Figure # is indicated below the featured microscopy system. The terminology follows ISO 10934:2020, “Microscopes—Vocabulary for light microscopy” (https://www.iso.org/standard/77327.html).

Figure 2

Effects of chemical fixation on the fluorescence of nuclear-targeted FPs. Time-lapse imaging of HeLa cells expressing H2B-StayGold, H2B-EGFP, H2B-mClover3 and H2B-mNeonGreen in HBSS. left, Representative low-magnification fluorescence images before fixation. Scale bars, 50 µm. right, Fluorescence intensities (FIs) of several cells are individually plotted against time. Within several hours after washing with HBSS, FIs of recovered fluorescence reached their plateaus. See Supplementary Fig. S1 for the mNeonGreen fluorescence.

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.

Figure 4

Photostability of StayGold, EGFP, mClover3 and mNeonGreen fused to H2B in living HeLa cells in HBSS with single-beam LSCM. (a) PSF_ill in the xy plane, calculated with NA = 0.95 and λ = 488 nm. The intensity profile (solid line) through the PSF_ill center is shown below. The Airy disk diameter is 0.626 µm. The PSF_ill section is compared to a square with a side length of 0.284 µm (dotted line). (b) Pixel sizes in the settings of the photobleaching experiments. top, zoom factor: × 1; pixel array size: 512 × 512. bottom, zoom factor: × 2; pixel array size: 1024 × 1024. (c) Scan speeds are indicated above the traces in units of µs/pixel. top, Only one illumination pulse was applied during the acquisition of each frame. bottom, Multiple illumination pulses were applied during the acquisition of each frame. The pulse widths are indicated below the traces with the calculated transit times. The traces are not drawn to scale. (d) Comparison of the photostability of the four green-emitting FPs in living cells in each experiment using single-beam LSCM. The scan-averaged irradiance value is shown at the upper right. 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).

Figure 5

Photostability of StayGold, EGFP, mClover3 and mNeonGreen fused to H2B in living HeLa cells in HBSS with multi-beam LSCM. Comparison of the photostability of the four green-emitting FPs in living cells in each experiment using multi-beam LSCM. The scan-averaged irradiance value and disk rotation speed are shown at the upper right. 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). The transit time is 3.1–8.7 µs (4000 rpm) or 8.3–23.2 µs (1500 rpm).