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.

Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature Imaging with a Genetically Encoded Nanothermometer

“…A recently reported FP, StayGold, is as bright as mNG, but shows remarkable photostability an order of magnitude higher than any prior FP. 57 Engineering ultraphotostable nanothermometers from such FPs could enable long-term temperature imaging with uncompromised speed and δT. Although we did not find apparent phototoxicity during the transient observation of intracellular heat diffusion, it should be considered toward longterm kilohertz imaging.…”

“…While this approach is effective for observing intracellular heat diffusion (reaching steady state in milliseconds) and other transient thermal events, long-term kilohertz imaging will require improved photostability. A recently reported FP, StayGold, is as bright as mNG, but shows remarkable photostability an order of magnitude higher than any prior FP . Engineering ultraphotostable nanothermometers from such FPs could enable long-term temperature imaging with uncompromised speed and δ T .…”

Real-time denoising enables high-sensitivity fluorescence time-lapse imaging beyond the shot-noise limit

“…The causes of this photon-limited challenge are manifold. First, the low photon yield of fluorescent indicators and their low concentration in labeled cells result in a lack of photons at the source 11 . Second, although using higher excitation power is a straightforward way to increase fluorescent photons, living systems are too fragile to tolerate high excitation dosage.…”

“…Comprehensive efforts have been invested to increase the photon budget of fluorescence microscopy, from designing high-performance fluorophores 11,[24][25][26] to upgrading the excitation and detection physics 20,[27][28][29] and developing data-driven denoising algorithms 23,[30][31][32][33] . We previously developed DeepCAD, a deep self-supervised denoising method for calcium imaging data, which effectively suppresses the detection noise and improves imaging SNR more than tenfold without requiring any high-SNR observations 33 .…”

Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa

“…However, the vast majority of these proteins have been optimized for animal systems for which the intracellular milieu and even amino acid codon preferences may be different from those in fungi; additionally, reported values for various protein characteristics including brightness, aggregation, pKa, etc. have been determined in vitro or are calculated from theoretical values (Campbell et al, 2020; Day and Davidson, 2009; Hirano et al, 2022). In fungi, these proteins can perform quite differently than how one might expect from the reported parameters.…”