A highly photostable and bright green fluorescent protein

Abstract

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

Fig. 1. Photostable properties of jellyfish-derived fluorescent protein StayGold.

a–c, Natural fluorescence of C. uchidae. Fluorescent polyps on a shell of N. livescens; the shell is ellipsoidal and measures approximately 1.5 cm by 1.0 cm (a). Fluorescence images of an isolated polyp (b) and a female medusa (c) superimposed on differential interference contrast images. Scale bars, 0.5 mm. d, Normalized excitation (dotted line) and emission (solid line) spectra of a tissue homogenate prepared from C. uchidae medusae. e, Amino acid sequence alignments of CU17S, StayGold, EGFP and DsRed. Residues whose side chains form the interior of the β-barrel are shaded (EGFP and DsRed). Asterisks: residues responsible for chromophore synthesis. The V168A mutation is indicated in red. f, Absorption spectra of CU17S (dotted line) and StayGold (solid line), normalized against the peak at 280 nm. g, Normalized excitation (dotted line) and emission (solid line) spectra of CU17S and StayGold. h, i, Photostability of green-emitting FPs under continuous WF illumination (5.6 W cm⁻²). Plotted as measured intensity versus time (top) or as intensity versus normalized total exposure time with an initial emission rate of 1,000 photons/s/molecule (bottom). h, Purified protein (1 μM) in polyacrylamide gel. The data are shared with Fig. 2b. i, Expressed in human (HeLa) cells in HBSS. j, k, Fluorescence images of StayGold-expressing and mNeonGreen-expressing (j) or EGFP-expressing (k) cells (stable HeLa cell transformants) at the indicated times (minutes:seconds). Illumination intensity: 16 W cm⁻². The gray scale indicates the lowest and highest intensities of the image. Scale bars, 20 μm. See Supplementary Video 1. l, log–log plot of bleaching half-time (Y) of StayGold (solid circles) or EGFP (open circles) and irradiance (X). Data were fitted to the equation log(Y) = −αlog(X) + c. α values were 0.90 and 0.96 for StayGold and EGFP, respectively. m, Chromophore maturation of StayGold (solid circles) and mNeonGreen (open circles) after cDNA transfection. Fluorescence intensity divided by the transfected cell occupation area was plotted every 6 hours and normalized to the maximum value. Data points are shown as means ± s.e.m. (n = 4 different experiments).

Fig. 2. Comparison of photobleaching curves.

a–c, Plotted as intensity versus normalized total exposure time, with an initial emission rate of 1,000 photons/s/molecule. See Supplementary Table 1. a, StayGold variants were photobleached under continuous WF illumination (5.6 W cm⁻²). b, Various colored FPs were photobleached under continuous WF illumination (3.4–5.6 W cm⁻²). Inset: A photoactivation component was noted for mOrange2 and mCardinal. All curves shown in Fig. 1h (top) are incorporated here. c, Multimeric FPs were photobleached under continuous WF illumination (3.4–5.6 W cm⁻²). The curve of StayGold shown in Fig. 1h is incorporated in a and b.

Fig. 3. Photostability of a cysteine mutant of StayGold targeted to the ER lumen.

HeLa cells expressing er-(n2)oxStayGold(c4) or er-oxGFP were subjected to continuous live imaging. Comparison was made side by side. Scale bars, 10 μm. a, WF (arc-lamp) illumination with an irradiance value of 0.21 W cm⁻². b, Spinning disk confocal illumination with an irradiance value of 3.5 W cm⁻². a,b, The first and last images are shown (top). The averaged fluorescence intensities of individual cells are plotted against time (bottom). c, Volumetric 3D-SIM imaging with an irradiance value of 2.4 W cm⁻². Repetitive collection of a 3D stack of 56 3D-SIM images. Raw and reconstructed 3D-SIM images of the 51st (left) or 48th (right) plane in z-series 1 and 2. The averaged fluorescence intensities of individual cells are plotted (bottom).

Fig. 4. Agonist-induced and antagonist-induced longitudinal changes in ER structures revealed by fast, sustainable, wide 3D-SIM.

a–d, Three neighboring HeLa cells expressing er-(n2)oxStayGold(c4) were imaged continuously at a temporal resolution of 2.6 frames per second. Illumination intensity: 2.4 W cm⁻². See Supplementary Video 3. a, Domain structure of er-(n2)oxStayGold(c4). SP(CRT), calreticulin signal peptide. b, A 3D-SIM image at one time point. c, FFT spectra of the first and last images (gray and black lines, respectively) from the reconstructed dataset. d, Quantification of ER network rearrangements. Temporal profiles of ER movement in individual cells are shown. e, In a separate experiment, Fluo3 was used to measure intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in cultured HeLa cells. In all observed cells, the application of histamine (10 μM) resulted in an initial peak and subsequent sinusoidal oscillations in [Ca²⁺]_i; the additional application of cyproheptadine (100 μM) stopped the oscillations, resulting in a drop in [Ca²⁺]_i to previous resting values. f–i, Three neighboring HeLa cells expressing er-(n2)oxStayGold were imaged continuously at 1.1 frames per second. Illumination intensity: 2.4 W cm⁻². See Supplementary Video 4. f, Domain structure of er-(n2)oxStayGold. g, A 3D-SIM image at one time point. h, FFT spectra for the first and last images (gray and black lines, respectively) of the reconstructed dataset. i, Quantification of ER network rearrangements. Temporal profiles of ER movement in individual cells. d,e,i, 10 μM histamine and 100 μM cyproheptadine were applied at 2 minutes and 4 minutes, respectively; the time zone of Ca²⁺ mobilization is shaded. Movement heat maps (d and i) or fluorescence images (e) at time points indicated by triangles, rhombuses and pentagons are shown. b,g, ER labeling shown in g was 8–9 times dimmer than that in b.c,h, L: low; H: high. Scale bars in b,d,e,g and i, 10 μm.

Fig. 5. Visualizing SARS-CoV-2 assembly in cells by StayGold SIM technology.

a, Domain structure of Nb(S1) = = StayGold. = =: EV linker, indicated as a gray bar. b, Absorption spectrum of Nb(S1) = = StayGold. Normalized against the peak at 280 nm. c, Coomassie brilliant blue staining for the visualization of Nb(S1) = = StayGold separated by SDS-PAGE. The migration position is indicated by an arrowhead. d, SPR experiment of S1 binding to immobilized Nb(S1) = = StayGold. Black traces show raw data; red lines show kinetic fit. e, VeroE6/TMPRSS2 cells infected with SARS-CoV-2 at an MOI of 0.1 and fixed at 24 hours post-infection (hpi). Spike protein (green). Nuclei were counterstained with Vybrant (magenta). Representative of n = 9 independent samples. f, SARS-CoV-2-infected (top) and SARS-CoV-2-uninfected (bottom) VeroE6/TMPRSS2 cells. Treated (left) and untreated (right) with Nb(S1) = = StayGold (3.4 μg ml⁻¹). Representative of n = 2 independent infections. g, A single-layer 3D-SIM image of SARS-CoV-2 spike protein (green) and Nuclear (blue) extracted from volumetric image data shown in Extended Data Fig. 5. Representative of n = 5 cells over three independent infections. h, A single-layer 3D-SIM image of SARS-CoV-2 spike protein (green), Nuclear (blue) and plasma membrane (red). Representative of n = 2 areas over two independent infections. i–k, Single-layer 3D-SIM images of SARS-CoV-2 spike protein (green) and ERGIC3 (magenta) (i), nsp8 (magenta) (j) or dsRNA (magenta) (k). Nuclear (blue) images are sectional but not SIM-reconstructed. Areas enclosed by white boxes are enlarged below. e,f, WF, wide-field microscopy observation. f–k, VeroE6/TMPRSS2 cells infected with SARS-CoV-2 (MOI = 0.02, 36 hpi). i–k, These single-layer 3D-SIM images are part of volumetric imaging data. Representative of n = 3 imaging experiments from a single sample per staining condition (Supplementary Fig. 15). g,h, Nuclear and plasma membrane images are sectional but not SIM-reconstructed. h,i, Samples are shared. Scale bars in e and f, 20 μm; g, 1 μm; and h–k, 5 μm. All the cells were fixed.

Extended Data Fig. 1. Analyzing rapid motion of ER tubules by using a new 3D-SIM technique that achieves nanoscale resolution on a millisecond time scale.

A COS-7 cell expressing er-(n2)oxStayGold(c4) was imaged by lattice SIM continuously at the temporal resolution of 134.47 frames/s for 5.473 s. The total number of frames was 736. See Supplementary Video 2. a, A binarized image. Scale bar, 10 μm. b, Oscillating ER tubules with relatively stable anchors were selected. Shown are three examples observed in boxed regions (ID: 0015, ID: 0018, and ID: 0023). top, High-magnification images. Scale bars, 1 μm. middle, Kymograph built along lines drawn perpendicularly to selected tubules. bottom, Fast Fourier transform (FFT) in a time window consisting of 128 (2⁷) consecutive time points (shaded zone). FFT spectra with a frequency unit of 1.05 (134.47/128) Hz and the Nyquist frequency (highest frequency to be analyzed) of 67.235 (134.47/2) Hz. In the selected peripheral ER, FFT spectra peaking at 4.2 and 5.25 Hz (asterisks) were detected. Shown is a representative of n = 5 independent experiments that imaged rapid motion of ER tubules at the temporal resolution of > 100 frames/s.

Extended Data Fig. 2. Cell-wide, spatiotemporally high-resolution, sustainable imaging of ER network rearrangement.

The three neighboring HeLa cells expressing er-(n2)oxStayGold(c4) (Fig. 4a–d) were analyzed. Scale bars, 5 μm. a, Magnified SIM and binarized images at t = 0 min (First frame) and t = 6 min (Last frame) of a peripheral region of the uppermost cell. Compare with Supplementary Fig. 11i. The preservation of 3D-SIM image quality in the last frame was confirmed in all six-minute experiments that employed er-(n2)oxStayGold(c4). Shown is a representative of n = 9 independent transfections, including experiments #1, #2, #3, and #4 (Supplementary Fig. 11a-d). b, Automatic quantification of ER network rearrangement. See Methods (Automatic quantification of ER network rearrangement). Magnifications of the boxed region are shown (bottom). Considering that the diameter of an ER tubule spanned several pixels, we reasoned that the sub-block size was optimal for the efficient detection of target movement.

Extended Data Fig. 3. Visualizing mitochondrial fission and fusion by StayGold SIM technology.

upper, HeLa cells stably expressing mt-(n1)StayGold were photographed by wide-field (WF) microscopy. The uniformity of fluorescence intensity among observed cells of this cell line is evident. lower, HeLa cells stably expressing mt-(n1)StayGold were imaged by 3D-SIM continuously with an irradiance value of 3.9 W/cm². Shown are two representative experimental results. In one experiment, which spanned 1 min 24.133 sec, Cell #22 was imaged at 4.4 Hz. In the other experiment, which spanned 1 min 27.014 sec, Cell #24 was imaged at 5.3 Hz. SIM images in boxed regions (ROIs) at the indicated times (min: sec) are presented to show fission (yellow arrowhead) and fusion (red arrowhead) events detected during the observation time periods. Each event is highlighted by a triad of images. Scale bars, 5 μm. Similar results were obtained from 2 other independent cultured cell samples.

Extended Data Fig. 4. Protein fusion applications.

a, Labeling microtubule plus-end with tdStayGold. left, Domain structure of EB3 = tdStayGold. EB3: Microtubule (MT)-associated end-binding protein 3. right, COS-7 cells expressing EB3 = tdStayGold were continuously imaged for 30 min (see Supplementary Video 6). Exposure time: 0.5 s. The first and last fluorescence images are shown. Comparison of the two images reveals that there was no substantial photobleaching during the 30-min observation. Scale bar, 20 μm. The EB3 dynamics shown are representative of n = 3 similar observations. b, Labeling postsynaptic density (PSD) with tdoxStayGold. top, Domain structure of PSD-95=tdoxStayGold. bottom, Cultured neurons prepared from embryonic rat hippocampus were cotransfected with the cDNAs of PSD-95=tdoxStayGold and mCherry at DIV (days in vitro) 5. Transfected neurons were imaged at DIV 25 in DMEM/F12 (1:1) containing 2% (vol/vol) FBS, N2-supplement (1×), and B-27 (1.5×) using the SpinSR10. Volumetric imaging was performed in z steps of 0.25 µm. The imaged volume comprised 30 z slices. Areas enclosed by white boxes are enlarged on the right side. Scale bars, 1 µm. Similar images were obtained from 6 other imaging experiments performed at DIV 21–25 on 4 independent neuronal samples. a,b, =: Coupler linker, a triple repeat of the amino acid linker Gly-Gly-Gly-Gly-Ser.

Extended Data Fig. 5. Volumetric 3D-SIM imaging of S protein in a SARS-CoV-2-infected VeroE6/TMPRSS2 cell.

The Nb(S1) = = StayGold fluorescence (green) was 3D-SIM reconstructed, whereas DAPI fluorescence (blue) was not. Images were acquired from z = 0 to z = 4.56 μm in steps of 0.12 μm upward (in the direction of a yellow arrow). A volume of 9.78 × 9.78 × 4.56 μm³ is 3D rendered. Two single-layer (xy) images at z = 1.44 and 3.24 μm are shown. Scale bar, 2 μm. The z position of the xy image of Fig. 5g is indicated. The volumetric imaging experiment in z steps of 0.12 μm was repeated 4 times with 39–79 slices. In each case, the fluorescence in the last frame (slice) was considerably strong for reliable super-resolution imaging.