volume | PIER Journals

Abstract

The second near-infrared region (NIR-II, 900-1880 nm) spectral window has garnered significant attention in bioimaging due to its moderate light absorption, diminished photon scattering and reduced autofluorescence. Exploiting NIR-II fluorescence, confocal microscopy has achieved deep in vivo imaging. In this study, we have identified that the fluorescence with wavelength beyond 1400 nm offers superior imaging quality for NIR-II confocal microscopy, irrespective of the laser excitation source being continuous-wave or pulsed. Furthermore, leveraging the multiphoton excitation capabilities of femtosecond laser, we have successfully integrated multiphoton excited visible fluorescence channels into the NIR-II fluorescence confocal microscopic system. We have successfully employed this novel system to acquire up to six distinct fluorescence microscopic imaging channels with negligible cross-channel interference, as well as multi-channel and large-depth in vivo observation of mouse brain and kidney.

1. Wang, Feifei, Yeteng Zhong, Oliver Bruns, Yongye Liang, and Hongjie Dai, "In vivo NIR-II fluorescence imaging for biology and medicine," Nature Photonics, 535-547, 2024. Google Scholar

2. Ramani, Rishi S., Ivy Tan, Lindsay Bussau, Christopher M. Angel, Michael McCullough, and Tami Yap, "Confocal microscopy in oral cancer and oral potentially malignant disorders: A systematic review," Oral Diseases, Vol. 29, No. 8, 3003-3015, 2023. Google Scholar

3. Teo, Alvin Wei Jun, Hassan Mansoor, Nigel Sim, Molly Tzu-Yu Lin, and Yu-Chi Liu, "In vivo confocal microscopy evaluation in patients with keratoconus," Journal of Clinical Medicine, Vol. 11, No. 2, 393, 2022. Google Scholar

4. Hong, Guosong, Shuo Diao, Junlei Chang, Alexander L. Antaris, Changxin Chen, Bo Zhang, Su Zhao, Dmitriy N. Atochin, Paul L. Huang, Katrin I. Andreasson, Calvin J. Kuo, and Hongjie Dai, "Through-skull fluorescence imaging of the brain in a new near-infrared window," Nature Photonics, Vol. 8, No. 9, 723-730, 2014. Google Scholar

5. Welsher, Kevin, Zhuang Liu, Sarah P. Sherlock, Joshua Tucker Robinson, Zhuo Chen, Dan Daranciang, and Hongjie Dai, "A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice," Nature Nanotechnology, Vol. 4, No. 11, 773-780, 2009. Google Scholar

6. Feng, Zhe, Tao Tang, Tianxiang Wu, Xiaoming Yu, Yuhuang Zhang, Meng Wang, Junyan Zheng, Yanyun Ying, Siyi Chen, Jing Zhou, et al. "Perfecting and extending the near-infrared imaging window," Light: Science & Applications, Vol. 10, No. 1, 197, 2021. Google Scholar

7. Yong, Ken-Tye, Jun Qian, Indrajit Roy, Hoon Hi Lee, Earl J. Bergey, Kenneth M. Tramposch, Sailing He, Mark T. Swihart, Anirban Maitra, and Paras N. Prasad, "Quantum rod bioconjugates as targeted probes for confocal and two-photon fluorescence imaging of cancer cells," Nano Letters, Vol. 7, No. 3, 761-765, 2007. Google Scholar

8. Chu, Liliang, Shaowei Wang, Kanghui Li, Wang Xi, Xinyuan Zhao, and Jun Qian, "Biocompatible near-infrared fluorescent nanoparticles for macro and microscopic in vivo functional bioimaging," Biomedical Optics Express, Vol. 5, No. 11, 4076-4088, 2014. Google Scholar

9. Sun, Chaowei, Yalun Wang, Hequn Zhang, and Jun Qian, "Near-infrared laser scanning confocal microscopy and its application in bioimaging," Optical and Quantum Electronics, Vol. 50, 1-9, 2018. Google Scholar

10. Alifu, Nuernisha, Abudureheman Zebibula, Ji Qi, Hequn Zhang, Chaowei Sun, Xiaoming Yu, Dingwei Xue, Jacky W. Y. Lam, Gonghui Li, Jun Qian, and Ben Zhong Tang, "Single-molecular near-infrared-II theranostic systems: Ultrastable aggregation-induced emission nanoparticles for long-term tracing and efficient photothermal therapy," ACS Nano, Vol. 12, No. 11, 11282-11293, 2018. Google Scholar

11. Yu, Wenbin, Bing Guo, Hequn Zhang, Jing Zhou, Xiaoming Yu, Liang Zhu, Dingwei Xue, Wen Liu, Xianhe Sun, and Jun Qian, "NIR-II fluorescence in vivo confocal microscopy with aggregation-induced emission dots," Science Bulletin, Vol. 64, No. 6, 410-416, 2019. Google Scholar

12. He, Mubin, Di Wu, Yuhuang Zhang, Xiaoxiao Fan, Siyi Zhuang, Wei Yang, Hui Lin, and Jun Qian, "Protein-enhanced NIR-IIb emission of indocyanine green for functional bioimaging," ACS Applied Bio Materials, Vol. 3, No. 12, 9126-9134, 2020. Google Scholar

13. Zhou, Jing, Tianxiang Wu, Runze Chen, Liang Zhu, Hequn Zhang, Yifei Li, Liying Chen, and Jun Qian, "Self-confocal NIR-II fluorescence microscopy for multifunctional in vivo imaging," Journal of Innovative Optical Health Sciences, Vol. 17, No. 1, 2350025, 2024. Google Scholar

14. Xia, Fei, Monique Gevers, Andreas Fognini, Aaron T. Mok, Bo Li, Najva Akbari, Iman Esmaeil Zadeh, Jessie Qin-Dregely, and Chris Xu, "Short-wave infrared confocal fluorescence imaging of deep mouse brain with a superconducting nanowire single-photon detector," ACS Photonics, Vol. 8, No. 9, 2800-2810, 2021. Google Scholar

15. Wang, Feifei, Fuqiang Ren, Zhuoran Ma, Liangqiong Qu, Ronan Gourgues, Chun Xu, Ani Baghdasaryan, Jiachen Li, Iman Esmaeil Zadeh, Johannes W. N. Los, Andreas Fognini, Jessie Qin-Dregely, and Hongjie Dai, "In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors," Nature Nanotechnology, Vol. 17, No. 6, 653-660, 2022. Google Scholar

16. Wu, Tianxiang, Yiwen Wang, Hui Lin, and Jun Qian, "A pervasive approach for determining the optimal tissue windows for near-infrared fluorescence imaging," Laser & Photonics Reviews, 2400628, 2024. Google Scholar

17. Bandi, Venu G., Michael P. Luciano, Mara Saccomano, Nimit L. Patel, Thomas S. Bischof, Jakob G. P. Lingg, Peter T. Tsrunchev, Meredith N. Nix, Bastian Ruehle, Chelsea Sanders, et al. "Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines," Nature Methods, Vol. 19, No. 3, 353-358, 2022. Google Scholar

18. Cosco, Emily D., Bernardo A. Arús, Anthony L. Spearman, Timothy L. Atallah, Irene Lim, Olivia S. Leland, Justin R. Caram, Thomas S. Bischof, Oliver T. Bruns, and Ellen M. Sletten, "Bright chromenylium polymethine dyes enable fast, four-color in vivo imaging with shortwave infrared detection," Journal of the American Chemical Society, Vol. 143, No. 18, 6836-6846, 2021. Google Scholar

19. Cosco, Emily D., Anthony L. Spearman, Shyam Ramakrishnan, Jakob G. P. Lingg, Mara Saccomano, Monica Pengshung, Bernardo A. Arús, Kelly C. Y. Wong, Sarah Glasl, Vasilis Ntziachristos, Martin Warmer, Ryan R. McLaughlin, Oliver T. Bruns, and Ellen M. Sletten, "Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time," Nature Chemistry, Vol. 12, No. 12, 1123-1130, 2020. Google Scholar

20. He, Yue, Shangfeng Wang, Peng Yu, Kui Yan, Jiang Ming, Chenzhi Yao, Zuyang He, Ahmed Mohamed El-Toni, Aslam Khan, Xinyan Zhu, Caixia Sun, Zuhai Lei, and Fan Zhang, "NIR-II cell endocytosis-activated fluorescent probes for in vivo high-contrast bioimaging diagnostics," Chemical Science, Vol. 12, No. 31, 10474-10482, 2021. Google Scholar

21. Lu, Lingfei, Benhao Li, Suwan Ding, Yong Fan, Shangfeng Wang, Caixia Sun, Mengyao Zhao, Chun-Xia Zhao, and Fan Zhang, "NIR-II bioluminescence for in vivo high contrast imaging and in situ ATP-mediated metastases tracing," Nature Communications, Vol. 11, No. 1, 4192, 2020. Google Scholar

22. Yang, Yanling, Yang Xie, and Fan Zhang, "Second near-infrared window fluorescence nanoprobes for deep-tissue in vivo multiplexed bioimaging," Advanced Drug Delivery Reviews, Vol. 193, 114697, 2023. Google Scholar

23. Yao, Chenzhi, Ying Chen, Mengyao Zhao, Shangfeng Wang, Bin Wu, Yiwei Yang, Dongrui Yin, Peng Yu, Hongxin Zhang, and Fan Zhang, "A bright, renal-clearable NIR-II brush macromolecular probe with long blood circulation time for kidney disease bioimaging," Angewandte Chemie, Vol. 134, No. 5, e202114273, 2022. Google Scholar

24. Gerdes, Hans-Hermann and Christoph Kaether, "Green fluorescent protein: Applications in cell biology," FEBS Letters, Vol. 389, No. 1, 44-47, 1996. Google Scholar

25. Gallo, Eugenio, "Fluorogen-activating proteins: Next-generation fluorescence probes for biological research," Bioconjugate Chemistry, Vol. 31, No. 1, 16-27, 2020. Google Scholar

26. Huang, Biao, Jun Hu, Hao Li, Meng-Yao Luo, Song Chen, Mingxi Zhang, Zhi-Jun Sun, and Ran Cui, "Near-infrared iib emitting nanoprobe for high-resolution real-time imaging-guided photothermal therapy triggering enhanced anti-tumor immunity," ACS Applied Bio Materials, Vol. 3, No. 3, 1636-1645, 2020. Google Scholar

27. Li, Hao, Meng Wang, Biao Huang, Su-Wen Zhu, Jun-Jie Zhou, De-Run Chen, Ran Cui, Mingxi Zhang, and Zhi-Jun Sun, "Theranostic near-infrared-iib emitting nanoprobes for promoting immunogenic radiotherapy and abscopal effects against cancer metastasis," Nature Communications, Vol. 12, No. 1, 7149, 2021. Google Scholar

28. Yu, Guang-Tao, Meng-Yao Luo, Hao Li, Song Chen, Biao Huang, Zhi-Jun Sun, Ran Cui, and Mingxi Zhang, "Molecular targeting nanoprobes with non-overlap emission in the second near-infrared window for in vivo two-color colocalization of immune cells," ACS Nano, Vol. 13, No. 11, 12830-12839, 2019. Google Scholar

29. Zhang, Mingxi, Jingying Yue, Ran Cui, Zhuoran Ma, Hao Wan, Feifei Wang, Shoujun Zhu, Ying Zhou, Yun Kuang, Yeteng Zhong, Dai-Wen Pang, and Hongjie Dai, "Bright quantum dots emitting at∼1,600 nm in the NIR-IIb window for deep tissue fluorescence imaging," Proceedings of the National Academy of Sciences, Vol. 115, No. 26, 6590-6595, 2018. Google Scholar

Dr. Tianxiang Wu

Dr. Weihang Geng

Dr. Yuhuang Zhang

Dr. Qiming Xia

Dr. Mingxi Zhang

Dr. Jin Li

Dr. Menglu Chen

Dr. Wang Xi

Dr. Shiyi Peng

Dr. Zhe Feng

Dr. Jun Qian