Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Silicon-RosIndolizine fluorophores with shortwave infrared absorption and emission profiles enable in vivo fluorescence imaging

Nature Chemistry volume 16, pages 970–978 (2024)Cite this article

Subjects

Abstract

In vivo fluorescence imaging in the shortwave infrared (SWIR, 1,000–1,700 nm) and extended SWIR (ESWIR, 1,700–2,700 nm) regions has tremendous potential for diagnostic imaging. Although image contrast has been shown to improve as longer wavelengths are accessed, the design and synthesis of organic fluorophores that emit in these regions is challenging. Here we synthesize a series of silicon-RosIndolizine (SiRos) fluorophores that exhibit peak emission wavelengths from 1,300–1,700 nm and emission onsets of 1,800–2,200 nm. We characterize the fluorophores photophysically (both steady-state and time-resolved), electrochemically and computationally using time-dependent density functional theory. Using two of the fluorophores (SiRos1300 and SiRos1550), we formulate nanoemulsions and use them for general systemic circulatory SWIR fluorescence imaging of the cardiovascular system in mice. These studies resulted in high-resolution SWIR images with well-defined vasculature visible throughout the entire circulatory system. This SiRos scaffold establishes design principles for generating long-wavelength emitting SWIR and ESWIR fluorophores.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Emission maxima of xanthene and silicon-substituted xanthene-based fluorophores.
Fig. 2: Full synthetic route to SiRos1300, SiRos1550 and SiRos1700.
Fig. 3: Molar absorptivity and normalized emission of SiRos1300, SiRos1550 and SiRos1700.
Fig. 4: Frontier molecular orbital analysis of the SiRos fluorophores.
Fig. 5: Preparation of SiRos nanoemulsions and visualization in vivo via intravenous injection.

Similar content being viewed by others

Data availability

The datasets generated during and/or analysed during the current study are available in a public repository https://zenodo.org/records/10079855 and are also available from the corresponding authors on reasonable request. Further graphical data pertaining to photophysical properties, electrochemistry, photoluminescent lifetimes, computation, micelle photostability, nanoemulsions, in vivo imaging, synthetic procedures and NMR spectra are also available in the Supplementary Information. Raw data for animal experiments can be accessed on the BioImage archive #S-BIAD548. Source Data are provided with this paper.

References

  1. Li, H., Wang, X., Li, X., Zeng, S. & Chen, G. Clearable shortwave-infrared-emitting NaErF4 nanoparticles for noninvasive dynamic vascular imaging. Chem. Mater. 32, 3365–3375 (2020).

    Article  CAS  Google Scholar 

  2. Barton, J. B., Demro, J. C., Gasparian, G. & Lange, M. Performance of an Uncooled Camera Utilizing an SWIR InGaAs 256 × 256 FPA for Imaging in the 1.0 µm – 1.7 µm Spectral Band. Defense Public Release: Technical Report, ADA399438 (US Department of Defense, 1998).

  3. Chinnathambi, S. & Shirahata, N. Recent advances on fluorescent biomarkers of near-infrared quantum dots for in vitro and in vivo imaging. Sci. Technol. Adv. Mater. 20, 337–355 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hong, G., Antaris, A. L. & Dai, H. Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng. 1, 0010 (2017).

    Article  CAS  Google Scholar 

  5. Owens, E. A., Henary, M., El Fakhri, G. & Choi, H. S. Tissue-specific near-infrared fluorescence imaging. Acc. Chem. Res. 49, 1731–1740 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tian, C. & Burgess, K. Flavylium and silylrhodapolymethines In excitation multiplexing. ChemPhotoChem 5, 702–704 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Usama, S. M. & Burgess, K. Hows and whys of tumor-seeking dyes. Acc. Chem. Res. 54, 2121–2131 (2021).

    Article  CAS  PubMed  Google Scholar 

  8. Wang, R., Li, X. & Yoon, J. Organelle-targeted photosensitizers for precision photodynamic therapy. ACS Appl. Mater. Interfaces 13, 19543–19571 (2021).

    Article  CAS  PubMed  Google Scholar 

  9. Liu, D. et al. Xanthene-based NIR-II dyes for in vivo dynamic imaging of blood circulation. J. Am. Chem. Soc. 17136–17143 (2021).

  10. Kim, B., Kim, H., Kim, S. & Hwang, Y. R. A brief review of non-invasive brain imaging technologies and the near-infrared optical bioimaging. Appl. Microsc. 51, 9 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wen, H. & Bellotti, E. Numerical study of the intrinsic recombination carriers lifetime in extended short-wavelength infrared detector materials: a comparison between InGaAs and HgCdTe. J. Appl. Phys. 119, 205702 (2016).

    Article  Google Scholar 

  12. Vittadello, L. et al. NIR–to–NIR Imaging: extended excitation up to 2.2 um using harmonic nanoparticles with a Tunable hIGh EneRgy (TIGER) widefield microscope. Nanomaterials 11, 3193 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sordillo, D. C., Sordillo, L. A., Sordillo, P. P., Shi, L. & Alfano, R. R. Short wavelength infrared optical windows for evaluation of benign and malignant tissues. J. Biomed. Opt. 22, 45002 (2017).

    Article  PubMed  Google Scholar 

  14. Carr, J. A. et al. Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared. Proc. Natl Acad. Sci. USA 115, 9080 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shapiro, A. et al. Tuning optical activity of IV–VI colloidal quantum dots in the short-wave infrared (SWIR) spectral regime. Chem. Mater. 28, 6409–6416 (2016).

    Article  CAS  Google Scholar 

  16. Shi, X. et al. Zn-doping enhances the photoluminescence and stability of PbS quantum dots for in vivo high-resolution imaging in the NIR-II window. Nano Res. 13, 2239–2245 (2020).

    Article  CAS  Google Scholar 

  17. Li, Y. et al. Novel NIR-II organic fluorophores for bioimaging beyond 1550 nm. Chem. Sci. 11, 2621–2626 (2020).

    Article  Google Scholar 

  18. Sun, C. et al. J-Aggregates of cyanine dye for NIR-II in vivo dynamic vascular imaging beyond 1500 nm. J. Am. Chem. Soc. 141, 19221–19225 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Carr, J. A. et al. Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green. Proc. Natl Acad. Sci. USA 115, 4465–4470 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Khan, Z. & Sekar, N. Far-red to NIR emitting xanthene-based fluorophores. Dyes Pigm. 208, 110735 (2022).

    Article  CAS  Google Scholar 

  21. Rathnamalala, C. S. L. et al. Donor–acceptor–donor NIR II emissive rhodindolizine dye synthesized by C–H bond functionalization. J. Org. Chem. 84, 13186–13193 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Chatterjee, S. et al. SWIR emissive RosIndolizine dyes with nanoencapsulation in water soluble dendrimers. RSC Adv. 11, 27832–27836 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chatterjee, S. et al. Design and synthesis of rhodIndolizine dyes with improved stability and shortwave infrared emission up to 1250 nm. J. Org. Chem. 87, 11319 (2022).

    Article  CAS  PubMed  Google Scholar 

  24. Grimm, J. B., Brown, T. A., Tkachuk, A. N. & Lavis, L. D. General synthetic method for Si-fluoresceins and Si-rhodamines. ACS Cent. Sci. 3, 975–985 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Best, Q. A., Sattenapally, N., Dyer, D. J., Scott, C. N. & McCarroll, M. E. pH−Dependent Si-fluorescein hypochlorous acid fluorescent probe: spirocycle ring-opening and excess hypochlorous acid-induced chlorination. J. Amer. Chem. Soc. 135, 13365–13370 (2013).

    Article  CAS  Google Scholar 

  26. Huang, Y. L., Walker, A. S. & Miller, E. W. A photostable silicon rhodamine platform for optical voltage sensing. J. Am. Chem. Soc. 137, 10767–10776 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zheng, Q. et al. Rational design of fluorogenic and spontaneously blinking labels for super-resolution Imaging. ACS Cent. Sci. 5, 1602–1613 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kushida, Y., Nagano, T. & Hanaoka, K. Silicon-substituted xanthene dyes and their applications in bioimaging. Analyst 140, 685–695 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Liu, M. H., Zhang, Z., Yang, Y. C. & Chan, Y. H. Polymethine-based semiconducting polymer dots with narrow-band emission and absorption/emission maxima at NIR-II for bioimaging. Angew. Chem. Int. Ed. 60, 983–989 (2021).

    Article  CAS  Google Scholar 

  30. Yang, Y. et al. Counterion-paired bright heptamethine fluorophores with NIR-II excitation and emission enable multiplexed biomedical imaging. Angew. Chem. Int. Ed. 61, e202117436 (2022).

    Article  CAS  Google Scholar 

  31. Ando, N., Soutome, H. & Yamaguchi, S. Near-infrared fluorescein dyes containing a tricoordinate boron atom. Chem. Sci. 10, 7816–7821 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lei, Z. et al. Synthesis of sterically protected xanthene dyes with bulky groups at C-3′ and C-7′. J. Org. Chem. 80, 11538–11543 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. McNamara, L. E. et al. Indolizine-squaraines: NIR fluorescent materials with molecularly engineered Stokes shifts. Chem. Eur. J. 23, 12494–12501 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Ndaleh, D. et al. Shortwave infrared absorptive and emissive pentamethine-bridged indolizine cyanine dyes. J. Org. Chem. 86, 15376–15386 (2021).

    Article  CAS  PubMed  Google Scholar 

  35. Deng, F. & Xu, Z. Heteroatom-substituted rhodamine dyes: structure and spectroscopic properties. Chin. Chem. Lett. 30, 1667–1681 (2019).

    Article  CAS  Google Scholar 

  36. Waggener, W. C. Absorbance of liquid water and deuterium oxide between 0.6 and 1.8 microns comparison of absorbance and effect of temperature. Anal. Chem. 30, 1569–1570 (1958).

    Article  CAS  Google Scholar 

  37. Wang, S. et al. Anti-quenching NIR-II molecular fluorophores for in vivo high-contrast imaging and pH sensing. Nat. Commun. 10, 1058 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Cosco, E. D. et al. Flavylium polymethine fluorophores for near- and shortwave infrared imaging. Angew. Chem. Int. Ed. 56, 13126–13129 (2017).

    Article  CAS  Google Scholar 

  39. Semonin, O. E. et al. Absolute photoluminescence quantum yields of IR-26 Dye, PbS, and PbSe quantum dots. J. Phys. Chem. Lett. 1, 2445–2450 (2010).

    Article  CAS  Google Scholar 

  40. Kratkiewicz, K., Manwar, R., Zhou, Y., Mozaffarzadeh, M. & Avanaki, K. Technical considerations in the Verasonics research ultrasound platform for developing a photoacoustic imaging system. Biomed. Opt. Express 12, 1050–1084 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Friedman, H. C. et al. Establishing design principles for emissive organic SWIR chromophores from energy gap laws. Chem 7, 3359–3376 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Atallah, T. L. et al. Decay-associated Fourier spectroscopy: visible to shortwave infrared time-resolved photoluminescence. Spectra. J. Phys. Chem. A 123, 6792–6798 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Caspar, J. V., Sullivan, B. P., Kober, E. M. & Meyer, T. J. Application of the energy gap law to the decay of charge transfer excited-states. Solvent effects. Chem. Phys. Lett. 91, 91–95 (1982).

    Article  CAS  Google Scholar 

  44. Siebrand, W. Radiationless transitions in polyatomic molecules. I. Calculation of Franck–Condon factors. J. Chem. Phys. 46, 440–447 (1967).

    Article  CAS  Google Scholar 

  45. Siebrand, W. Radiationless transitions in polyatomic molecules. II. Triplet-ground-state transitions in aromatic hydrocarbons. J. Chem. Phys. 47, 2411–2422 (1967).

    Article  CAS  Google Scholar 

  46. Caspar, J. V. & Meyer, T. J. Application of the energy gap law to nonradiative, excited-state decay. J. Phys. Chem. 87, 952–957 (1983).

    Article  CAS  Google Scholar 

  47. Maillard, J. et al. Universal quenching of common fluorescent probes by water and alcohols. Chem. Sci. 12, 1352–1362 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Zheng, L., Polizzi, N. F., Dave, A. R., Migliore, A. & Beratan, D. N. Where Is the electronic oscillator strength? Mapping oscillator strength across molecular absorption. Spectra. J. Phys. Chem. A 120, 1933–1943 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Frisch, M. J., Pople, J. A. & Binkley, J. S. Self-consistent molecular orbital methods 25. Supplementary functions for Gaussian basis sets. J. Chem. Phys. 80, 3265–3269 (1983).

    Article  Google Scholar 

  50. Lee, C., Yang, W. & Parr, R. G. Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785–789 (1988).

    Article  CAS  Google Scholar 

  51. Becke, A. D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1993).

    Article  CAS  Google Scholar 

  52. Frisch, M. J. et al. Gaussian 16, Revision A.03 (Gaussian, Inc, 2016).

  53. Day, R. A., Estabrook, D. A., Logan, J. K. & Sletten, E. M. Fluorous photosensitizers enhance photodynamic therapy with perfluorocarbon nanoemulsions. Chem. Commun. 53, 13043–13046 (2017).

    Article  CAS  Google Scholar 

  54. Cosco, E. D. et al. Bright chromenylium polymethine dyes enable fast, four-color in vivo imaging with shortwave infrared detection. J. Am. Chem. Soc. 143, 6836–6846 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Flock, S. T., Jacques, S. L., Wilson, B. C., Star, W. M. & van Gemert, M. J. Optical properties of Intralipid: a phantom medium for light propagation studies. Lasers Surg Med 12, 510–519 (1992).

    Article  CAS  PubMed  Google Scholar 

  56. Wang, F. et al. In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors. Nat. Nanotechnol. 17, 653–660 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Rurack, K. & Spieles, M. Fluorescence quantum yields of a series of red and near-infrared dyes emitting at 600–1000 nm. Anal. Chem. 83, 1232–1242 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Lu, L. et al. NIR-II bioluminescence for in vivo high contrast imaging and in situ ATP-mediated metastases tracing. Nat. Commun. 11, 4192 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Ishchenko, A. A. et al. Electronic structure and spectral-fluorescent properties of thiopyrylo-4-tricarbocyanine laser dyes. Opt. Spectrosc. 129, 926–934 (2021).

    Article  CAS  Google Scholar 

  60. Pengshung, M. et al. Silicon incorporation in polymethine dyes. Chem. Commun. 56, 6110–6113 (2020).

    Article  CAS  Google Scholar 

  61. Soleymani, J., Zamani-Kalajahi, M., Ghasemi, B., Kenndler, E. & Jouyban, A. Solubility of sodium acetate in binary mixtures of methanol, 1-propanol, acetonitrile, and water at 298.2 K. J. Chem. Eng. Data 58, 3399–3404 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the National Science Foundation (NSF; award no. 1757220 to W.E.M., D.N., A.K.S., N.I.H. and J.H.D.), the National Institute of Health (NIH; award no. 1R01EB027172 to E.M.S.), the Tobacco-Related Disease Research Program (TRDRP; award no. T32DT4847 to E.Y.L.), and the UCLA for the Dissertation Year Fellowship (I.L.) for their financial support. J.R.C. and H.C.F. would like to acknowledge National Science Foundation Career Award No. 1945572 and support from the Cottrell Award. This manuscript is based on work supported by the National Science Foundation Graduate Research Fellowship Program awarded to W.E.M. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We would also like to acknowledge the late Douglas Hamm from HORIBA Scientific for his assistance in facilitating the fluorescence emission data acquisition in this study.

Author information

Author notes

  1. These authors contributed equally: William E. Meador, Eric Y. Lin.

Authors and Affiliations

  1. University of Mississippi, Department of Chemistry and Biochemistry, Oxford, MS, USA

    William E. Meador, David Ndaleh, Abdul K. Shaik, Nathan I. Hammer & Jared H. Delcamp

  2. University of California Los Angeles, Department of Chemistry and Biochemistry, Los Angeles, CA, USA

    Eric Y. Lin, Irene Lim, Hannah C. Friedman, Justin R. Caram & Ellen M. Sletten

  3. HORIBA Scientific, Piscataway, NJ, USA

    Boqian Yang

  4. Air Force Research Laboratory, Materials and Manufacturing Directorate (RXNC), Wright-Patterson AFB, Dayton, OH, USA

    Jared H. Delcamp

Authors
  1. William E. Meador
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric Y. Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Irene Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hannah C. Friedman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David Ndaleh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Abdul K. Shaik
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nathan I. Hammer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Boqian Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Justin R. Caram
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ellen M. Sletten
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jared H. Delcamp
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.E.M. synthesized the intermediates and fluorophores herein and collected absorption, emission, cyclic voltammetry measurements of the fluorophores along with the computational data. E.Y.L. and I.L. conducted the in vivo and capillary imaging experiments. H.C.F. conducted the photoluminescent lifetime experiments. D.N. synthesized compound 6. A.K.S. collected the emission spectrum and calculated the fluorescence quantum yield of SiRos1300. N.I.H. designed and supervised the fluorescence studies. B.Y. collected the fluorescence emission spectra for all fluorophores at HORIBA Scientific. J.R.C. designed and supervised the photoluminescent lifetime experiments. E.M.S. designed and supervised the in vivo and capillary imaging experiments. J.H.D. designed and supervised the design, synthesis and characterization of the fluorophores herein. W.E.M. wrote the initial draft of the manuscript and all authors assisted in editing subsequent drafts. All authors discussed the results and contributed to the paper.

Corresponding authors

Correspondence to Ellen M. Sletten or Jared H. Delcamp.

Ethics declarations

Competing interests

W.E.M., D.N. and J.H.D. have a patent pending which includes the dyes studied herein, US Patent 20220370641A1. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks Luke Lavis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Evaluation and comparison of SiRos1300 and SiRos1550 depth and resolution in 1% Intralipid relative to shorter wavelength SWIR-emitting dyes.

(A) Schematic of 1% Intralipid depth experiment. All dyes were initially brightness-matched in CH2Cl2 on the SWIR camera and subsequently imaged at 0.5 mm depth increments using a 1,300, 1,400, or 1,500 nm LP filter. (B) Intensity plots at each 0.5 mm depth normalized to the brightest dye when using a 1,300, 1,400, or 1,500 nm LP filter. Note that the increased baseline in the 1,300 LP images is due to stray excitation light.

Source data

Supplementary information

Supplementary Information

Supplementary figures, tables, discussion and chemical characterization data.

Reporting Summary

Source data

Source Data Fig. 3

Molar absorptivity and normalized and corrected emission spectra in dichloromethane.

Source Data Fig. 5

Absorption spectra of fluorophores in the nanoemulsions, cross-sectional intensities from mouse imaging experiments, and capillary brightness.

Source Data Extended Data Fig. 1

Capillary brightness data from depth penetration experiments.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Meador, W.E., Lin, E.Y., Lim, I. et al. Silicon-RosIndolizine fluorophores with shortwave infrared absorption and emission profiles enable in vivo fluorescence imaging. Nat. Chem. 16, 970–978 (2024). https://doi.org/10.1038/s41557-024-01464-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-024-01464-6

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing