Jihwan “James” Lee is a temporary post-doctoral associate at the St-Pierre Lab, Department of Neuroscience, Baylor College of Medicine, Rice University. Advisor: Dr. François St-Pierre. His research focuses on engineering bright, photostable fluorescent proteins for extended live-cell imaging using high-throughput single-cell screening. His work uses Mightex’s Polygon1000 Pattern Illuminator.

Bright and Photostable Yellow Fluorescent Proteins for Extended Imaging(Mightex Travel Award, ASCB 2024)

Introduction

Metagenomic screening and protein engineering have produced a broad color palette of fluorescent proteins (FPs) to visualize biological events and structures. However, many FPs rapidly photobleach under repeated or prolonged illumination, limiting the duration and temporal resolution in live cell microscopy. Rapid photobleaching is especially pronounced in high-power and long-exposure techniques such as super-resolution¹, single-molecule^2,3, and voltage imaging⁴. StayGold, a highly photostable green FP reported recently, facilitates prolonged imaging experiments^5–8. However, the photolability of FPs in other spectral ranges persists, limiting extended multi-spectral imaging. Notably, yellow FPs (YFPs) typically photobleach faster than counterparts from other spectral classes. Given the widespread use of YFPs in fluorescent biosensors^9–11 and multi-spectral imaging^12–14, enhancing their photostability is imperative.

Results

Single-cell screening platform identifies two bright YFPs with improved photostability

We sought to address the relative photolability of current YFPs via high-throughput protein engineering. Screening was performed using SPOTlight, our pooled single-cell screening platform¹⁵ (Fig. 1). We randomly mutagenized mGold¹⁵, the most photostable monomeric YFP we previously derived from mVenus¹⁵ (Fig. 1a, step 1). We screened the resulting libraries in yeast cells, evaluating the brightness and photostability of 1,125,438 cells representing 204,987 variants across 7 rounds of screening (Fig. 1a, step 2). In each screening round, we optically labeled 200-400 single variants (Fig. 1a, step 3→4) for retrieval via Fluorescence Activated Cell Sorting (FACS). Using the Mightex Polygon 1000 digital micromirror device was critical because it enabled precise single-cell optical labeling. Compared to DMD from another company, Mightex’s DMD provided greater illumination strength on the sample plane, enabling faster optical tagging. Optically labeled cells were then grown into colonies on agar plates, picked, expanded, and plated onto multi-well plates (Fig. 1a, step 4). We performed population-level validation by quantifying the mean brightness and photostability of thousands of cells per variant, and the top 5-10 variants were sequenced.

Characterization and application of mGold2s and mGold2t

The two best variants based on brightness and photostability were named mGold2s and mGold2t. When we imaged live mammalian cells expressing mGold2s and mGold2t using continuous widefield illumination of 520/5 nm (central wavelength/spectral width) light at 2.4 mW/mm² for 1 hour, mGold2s and mGold2t retained 50% and 43% of their original fluorescence, corresponding to photobleaching half-lives of 60 and 42 minutes, respectively (Fig. 1b). In contrast, mVenus and mGold rapidly photobleached with half-lives of 3 and 12 minutes, respectively. mGold2s and mGold2t showed different photobleaching patterns: mGold2t initially photobleached more rapidly than mGold2s, but this trend reversed at later time points (Fig. 1c).

Figure 1. Development, characterization, and application of mGold2 YFPs. (a) SPOTlight screening workflow used to develop bright and photostable YFP variants. (b) mGold2s and mGold2t expressed in HEK293A cells retained around half of their fluorescence after continuous illumination of 520/5-nm light at 2.4 mW/mm² for 1 hr. (c) YFP-LifeAct fusions were expressed in COS-7 cells to visualize actin filaments. (d) Imaging workflow. (e) mGold2s and mGold2t showed greater photostability than mVenus under TIRF-SIM with 488 nm light at 95 mW/mm². Bottom graph: Traces denoted the mean, and the thin shaded area are the 95% CI. n = 40-50 regions of interest across 10 cells. (f) mGold2s enabled extended visualization of actin dynamics. The arrow denotes an actin blip.

To demonstrate the utility of the photostable mGold2 variants, we assessed the performance of mGold2s and mGold2t in super-resolution imaging using structure-illuminated microscopy (SIM). We imaged actin filaments in COS-7 cells expressing YFP-LifeAct fusions at a rate of 9 Hz for ~1.5 minutes, using a 488-nm laser at 95 mW/mm². Each time point comprised nine raw frames reconstructed into a single SIM image. The resolution of SIM images was further enhanced through sparse deconvolution¹⁶ (Fig. 1c–d). mGold2s and mGold2t maintained over 80% of their initial fluorescence, significantly outperforming mVenus, which retained only about 25% (Fig. 2e). mGold2s enabled visualization of actin dynamics similar to the “actin blip” dynamics described previously¹⁷ (Fig. 2f). This dynamic event involves actin punctum formation at the network node followed by disassembly of the punctum and can be detected as a spike in fluorescence intensity. These dynamics could not be observed using photolabile mVenus because of rapid photobleaching.

Conclusion

We anticipate that these new variants will set a new gold standard in the realm of monomeric YFPs, offering extended imaging durations and improved temporal resolution for biological research and biotechnological applications.

References

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2. Schirripa Spagnolo, C. & Luin, S. Choosing the Probe for Single-Molecule Fluorescence Microscopy. Int. J. Mol. Sci. 23, 14949 (2022).

3. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

4. Yang, H. H. & St-Pierre, F. Genetically Encoded Voltage Indicators: Opportunities and Challenges. J. Neurosci. 36, 9977–9989 (2016).

5. Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat. Biotechnol. 40, 1132–1142 (2022).

6. Ando, R. et al. StayGold variants for molecular fusion and membrane-targeting applications. Nat. Methods (2023) doi:10.1038/s41592-023-02085-6.

7. Ivorra-Molla, E. et al. A monomeric StayGold fluorescent protein. Nat. Biotechnol. (2023) doi:10.1038/s41587-023-02018-w.

8. Zhang, H. et al. Bright and stable monomeric green fluorescent protein derived from StayGold. Nat. Methods (2024) doi:10.1038/s41592-024-02203-y.

9. Tutol, J. N. et al. Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells. ACS Cent. Sci. 10, 77–86 (2024).

10. Ni, Q. et al. Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit. Nat. Chem. Biol. 7, 34–40 (2011).

11. Ponsioen, B. et al. Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep. 5, 1176–1180 (2004).

12. Cohen, S., Valm, A. M. & Lippincott-Schwartz, J. Multispectral live-cell imaging. Curr. Protoc. Cell Biol. 79, e46 (2018).

13. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

14. Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W. & Sanes, J. R. Improved tools for the Brainbow toolbox. Nat. Methods 10, 540–547 (2013).

15. Lee, J. et al. Versatile phenotype-activated cell sorting. Sci. Adv. 6, eabb7438 (2020).

16. Zhao, W. et al. Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy. Nat. Biotechnol. 40, 606–617 (2022).

17. Mo, Y. et al. Quantitative structured illumination microscopy via a physical model-based background filtering algorithm reveals actin dynamics. Nat. Commun. 14, 3089 (2023).

Illuminating New Frontiers in Cell Signaling with Mightex Polygon: Photocaged L-Lactate and Enhanced Fluorescent Imaging(Nature Communications, 2025 & Communications Chemistry, 2025)

Recent breakthroughs in optogenetics and live-cell imaging have unlocked exciting possibilities for probing cellular processes with exquisite spatiotemporal control. Two new studies — one focused on photoreleasable metabolic tools and another on advanced fluorescent proteins — showcase the power of photoactivation, with the Mightex Polygon digital micromirror device (DMD) playing a critical role in precise targeted illumination.

In the first study by Miyazaki et al. (Communications Chemistry, 2025), researchers synthesized a novel photocaged version of L-lactate (MNI-L-lac) to explore this important molecule’s signaling and metabolic roles. Long dismissed as a metabolic byproduct, L-lactate is now recognized for its signaling functions, including modulation of the GPR81 receptor and regulation of cAMP levels. The authors demonstrated that upon light activation, MNI-L-lac efficiently releases L-lactate, which then engages both extracellular and intracellular biosensors (eLACCO2.1 and R-iLACCO1.2, respectively, Figure 1). Notably, photoactivation was performed using a 405 nm light pulse delivered via the Mightex Polygon, allowing for spatially resolved uncaging during real-time imaging of cellular responses.

Figure 1: Uncaging of MNI-L-lac with the Mightex Polygon using 405nm illumination. L-lactate biosensor reports increased intracellular concentration after uncaging.

In parallel, Lee et al. (Nature Communications, 2025) tackled a longstanding limitation in live-cell imaging: the rapid photobleaching of yellow fluorescent proteins (YFPs). They engineered mGold2s and mGold2t — YFPs with dramatically enhanced photostability (up to 25× that of mVenus) while maintaining brightness, all through a high-throughput single-cell screening enabled by the Mightex Polygon.

Figure 2: High-throughput single-cell screening used to engineer more photostable YFP constructs. The Mightex Polygon DMD optically tags resistant cells.

Together, these studies exemplify the synergy between chemical biology and optical engineering. In the first study, precise targeting of light was essential to control when and where L-lactate was released. In the second study, the Mightex Polygon restricted light to a 4 μm × 4 μm region centered on the target cells.

In summary, these innovations expand the toolkit for probing cell physiology and underscore the Mightex Polygon’s growing role in modern optobiology labs.

To read the full publication, please click here (Lee et al.) or here (Miyazaki et al.).