Engineered Multicolor NIR-I/II Fluorescent Proteins for Precise In Vivo Visualization and Imaging-Guided Surgery

Abstract

Even though existing near-infrared probes have successfully achieved deep tissue imaging with high contrast, it remains challenging to image many species simultaneously under complicated biological conditions. We rationally design and synthesize a series of nonamethine dyes featuring a unique double meso-Cl structure, which can covalently bind to proteins, resulting in significant fluorescence enhancement, thereby further filling a critical gap of multicolor fluorescent proteins (FPs) in 915 nm channel bioimaging. NIR-940 exhibits distinct binding sites and modes with HSA compared to heptamethine dyes, which likely contributes to its high albumin binding efficiency. Given that FPs enhance fluorescence emission of the chromophore and are entirely dependent on the metabolic behavior of the host protein, this approach allows for the development of customized FPs tailored to specific imaging needs—an advantage unmatched by other NIR-I/II contrast agents. By selecting various NIR-I and NIR-II chromophores with spectrally distinguishable emissions, we construct a multi-channel imaging platform based entirely on FPs and validated its robustness and versatility in monitoring various physiological states across multiple local and systemic imaging applications, including the mesentery, retroperitoneal region, and stroke-affected mouse brain.

Keywords

endogenous targeting agent / engineered fluorescent proteins / imaging-guided surgery / multicolor NIR imaging / rationally designed nonamethine dyes

References

[1] F. Wang, Y. Zhong, O. Bruns, Y. Liang, and H. Dai, “In Vivo NIR-II Fluorescence Imaging for Biology and Medicine,” Nature Photonics 18, no. 6 (2024): 535–547.

[2] E. L. Schmidt, Z. Ou, E. Ximendes, et al., “Near-Infrared II Fluorescence Imaging,” Nature Reviews Methods Primers 4, no. 1 (2024): 23.

[3] K. Welsher, Z. Liu, S. P. Sherlock, et al., “A Route to Brightly Fluorescent Carbon Nanotubes for Near-Infrared Imaging in Mice,” Nature Nanotechnology 4, no. 11 (2009): 773–780.

[4] Z. Hu, C. Fang, B. Li, et al., “First-In-Human Liver-Tumour Surgery Guided by Multispectral Fluorescence Imaging in the Visible and Near-Infrared-I/II Windows,” Nature Biomedical Engineering 4, no. 3 (2020): 259–271.

[5] Z. Lei and F. Zhang, “Molecular Engineering of NIR-II Fluorophores for Improved Biomedical Detection,” Angewandte Chemie International Edition 60, no. 30 (2021): 16294–16308.

[6] S. Zhu, R. Tian, A. L. Antaris, X. Chen, and H. Dai, “Near-Infrared-II Molecular Dyes for Cancer Imaging and Surgery,” Advanced Materials 31, no. 24 (2019): 1900321.

[7] H. Zhao, H. Zhao, Y. Jiao, et al., “Biosynthetic Molecular Imaging Probe for Tumor-Targeted Dual-Modal Fluorescence/Magnetic Resonance Imaging,” Biomaterials 256 (2020): 120220.

[8] P. Van Roessel and A. H. Brand, “Imaging Into the Future: Visualizing Gene Expression and Protein Interactions With Fluorescent Proteins,” Nature Cell Biology 4, no. 1 (2002): E15–E20.

[9] D. Zhang, Z. Chen, Z. Du, et al., “Design of a Palette of SNAP-Tag Mimics of Fluorescent Proteins and Their Use as Cell Reporters,” Cell Discovery 9, no. 1 (2023): 56.

[10] J. Kong, Y. Wang, W. Qi, M. Huang, R. Su, and Z. He, “Green Fluorescent Protein Inspired Fluorophores,” Advances in Colloid and Interface Science 285 (2020): 102286.

[11] R. N. Day and M. W. Davidson, “The Fluorescent Protein Palette: Tools for Cellular Imaging,” Chemical Society Reviews 38, no. 10 (2009): 2887–2921.

[12] Z. Chang, J. Cai, C. Liu, et al., “Strong Exciton Emission and Ultra-Photostable Near Infrared-II Fluorescent Protein for In Vivo Imaging,” Advanced Functional Materials 35, no. 9 (2024): 2416366.

[13] M. Chen, Z. Feng, X. Fan, et al., “Long-Term Monitoring of Intravital Biological Processes Using Fluorescent Protein-Assisted NIR-II Imaging,” Nature Communications 13, no. 1 (2022): 6643.

[14] D. M. Shcherbakova, O. V. Stepanenko, K. K. Turoverov, and V. V. Verkhusha, “Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics Across Scales,” Trends in Biotechnology 36, no. 12 (2018): 1230–1243.

[15] H. Zhang, G. D. Lesnov, O. M. Subach, et al., “Bright and Stable Monomeric Green Fluorescent Protein Derived From StayGold,” Nature Methods 21, no. 4 (2024): 657–665.

[16] O. S. Oliinyk, C. Ma, S. Pletnev, et al., “Deep-Tissue SWIR Imaging Using Rationally Designed Small Red-Shifted Near-Infrared Fluorescent Protein,” Nature Methods 20, no. 1 (2023): 70–74.

[17] C. Li, A. G. Tebo, M. Thauvin, et al., “A Far-Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging,” Angewandte Chemie International Edition 59, no. 41 (2020): 17917–17923.

[18] D. S. Bindels, L. Haarbosch, L. Van Weeren, et al., “mScarlet: A Bright Monomeric Red Fluorescent Protein for Cellular Imaging,” Nature Methods 14, no. 1 (2017): 53–56.

[19] A. Sakaue-Sawano, H. Kurokawa, T. Morimura, et al., “Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression,” Cell 132, no. 3 (2008): 487–498.

[20] N. C. Shaner, M. Z. Lin, M. R. Mckeown, et al., “Improving the Photostability of Bright Monomeric Orange and Red Fluorescent Proteins,” Nature Methods 5, no. 6 (2008): 545–551.

[21] J. Xu, N. Zhu, Y. Du, et al., “Biomimetic NIR-II Fluorescent Proteins Created From Chemogenic Protein-Seeking Dyes for Multicolor Deep-Tissue Bioimaging,” Nature Communications 15, no. 1 (2024): 2845.

[22] O. E. Semonin, J. C. Johnson, J. M. Luther, A. G. Midgett, A. J. Nozik, and M. C. Beard, “Absolute Photoluminescence Quantum Yields of IR-26 Dye, PbS, and PbSe Quantum Dots,” Journal of Physical Chemistry Letters 1, no. 16 (2010): 2445–2450.

[23] A. B. Uzdensky, “Photothrombotic Stroke as a Model of Ischemic Stroke,” Translational Stroke Research 9, no. 5 (2018): 437–451.

[24] M. J. Frisch, G. W. Trucks, H. B. Schlegel, et al., Gaussian 16, Revision D.01 (Gaussian Inc., 2016).

[25] J. Cao, Q. Ren, F. Chen, and T. Lu, “Comparative Study on the Methods for Predicting the Reactive Site of Nucleophilic Reaction,” Science China Chemistry 58, no. 12 (2015): 1845–1852.

[26] Y. Xu, C. Yang, Y. Wu, et al., “In Situ Albumin-Hitchhiking NIR-II Probes for Accurate Detection of Micrometastases,” Nano Letters 23, no. 12 (2023): 5731–5737.

[27] J. C. Coffey, K. G. Byrnes, D. J. Walsh, and R. M. Cunningham, “Update on the Mesentery: Structure, Function, and Role in Disease,” Lancet Gastroenterology & Hepatology 7, no. 1 (2022): 96–106.

[28] S. H. Emile, “Refining the Anatomy of the Mesentery: How Can It Affect Management of Colorectal Cancer?,” Lancet Gastroenterology & Hepatology 2, no. 4 (2017): 244.

[29] A. Coffin, I. Boulay-Coletta, D. Sebbag-Sfez, and M. Zins, “Radioanatomy of the Retroperitoneal Space,” Diagnostic and Interventional Imaging 96, no. 2 (2015): 171–186.

[30] S. B. Hladky and M. A. Barrand, “The Glymphatic Hypothesis: The Theory and the Evidence,” Fluids and Barriers of the CNS 19, no. 1 (2022): 9.

[31] M. K. Rasmussen, H. Mestre, and M. Nedergaard, “The Glymphatic Pathway in Neurological Disorders,” Lancet Neurology 17, no. 11 (2018): 1016–1024.

[32] L. M. Hablitz, H. S. Vinitsky, Q. Sun, et al., “Increased Glymphatic Influx is Correlated With High EEG Delta Power and Low Heart Rate in Mice Under Anesthesia,” Science Advances 5, no. 2 (2019): eaav5447.

[33] H. Benveniste, H. Lee, F. Ding, et al., “Anesthesia With Dexmedetomidine and Low-Dose Isoflurane Increases Solute Transport via the Glymphatic Pathway in Rat Brain When Compared With High-Dose Isoflurane,” Anesthesiology 127, no. 6 (2017): 976–988.

[34] Y.-J. Wang, Y.-R. Sun, Y.-H. Pei, et al., “The Lymphatic Drainage Systems in the Brain: A Novel Target for Ischemic Stroke?,” Neural Regeneration Research 18, no. 3 (2023): 485.

[35] X. Jiang, A. V. Andjelkovic, L. Zhu, et al., “Blood-Brain Barrier Dysfunction and Recovery After Ischemic Stroke,” Progress in Neurobiology 163–164 (2018): 144–171.

[36] A. Kassner and Z. Merali, “Assessment of Blood–Brain Barrier Disruption in Stroke,” Stroke 46, no. 11 (2015): 3310–3315.

[37] Y. Du, J. Xu, X. Zheng, et al., “NIR-II Protein-Escaping Dyes Enable High-Contrast and Long-Term Prognosis Evaluation of Flap Transplantation,” Advanced Materials 36, no. 14 (2024): 2311515.

RIGHTS & PERMISSIONS

2025 The Author(s). SmartMat published by Tianjin University and John Wiley & Sons Australia, Ltd.