In addition, owing to the Adenosine Triphosphate (ATP)-dependent manner of NIR-II-BPs 42, intracellular ATP-mediated imaging in solid and metastatic tumors is performed with high tumor-to-normal tissue (T/N) ratio, demonstrating that the NIR-II-BPs may hold a great promise in metastases tracing. The flexibility of the energy transfer strategy also allows the emission wavelength of the bioluminescence probes to be tuned, which enables high-performance multicolor imaging on superficial vessels and tumor tissues simultaneously in a living mouse. Compared with NIR-II fluorescence imaging on vascular and lymphatic systems, strikingly high SNRs and improvement in spatial resolution are obtained by using NIR-II bioluminescence imaging. In this work, we report the development of NIR-II bioluminescence probes (NIR-II-BPs) based on a specially designed cyanine dye FD-1029 to overcome the above challenges by integrating a BRET process with a two-step fluorescence resonance energy transfer (FRET) process (Fig. However, the necessity of external illuminations for excitation in in vivo NIR-II fluorescence imaging inevitably leads to other unfavorable outcomes, such as the autofluorescence background, potential light-induced overheating effect and inhomogeneous illumination in wide-field imaging 39, 40, 41. One recent clinical study also highlights the promising clinical potential of intraoperative NIR-ΙΙ fluorescence imaging and NIR-II image-guided surgery 38. Compared with imaging in VIS and NIR-I window, recent works have revealed that optical imaging in the second near infrared (NIR-II, 1000–1700 nm) region can achieve higher spatial resolution in deep tissues (above 1 cm) because the above adverse factors are suppressed 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. Through bioluminescence resonance energy transfer (BRET) 16, the self-illuminating emission wavelengths in VIS reigon have successfully been extended to the first near-infrared (NIR-I, 700–900 nm) window with low tissue absorption for improved tumor and lymph node imaging as well as molecular imaging of enzyme activities 17, 18, 19, but the scattering effect remains an obstacle, blurring the image at larger penetration depth in tissue 20. However, the short emission wavelengths locating in the visible range (VIS, 400–700 nm) of conventional bioluminescence imaging bring about the intrinsic limitation of strong tissue absorption and scattering for in vivo imaging 15, which restricts the detection of signals from deep tissues with high sensitivity and high spatio-temporal resolution. So far, bioluminescence imaging has been widely used for tracking cells 10, monitoring gene expression 11, 12, sensing small bioactive molecules 13, and tumor imaging 14. Therefore, optical imaging strategies such as bioluminescence imaging, chemiluminescence imaging and afterglow imaging have attracted great interest, as they eliminate the demand for simultaneous light excitation. One of the dominant limitations against acquiring high-performance imaging with high signal-to-noise ratios (SNRs) is the autofluorescence background arising from endogenous fluorophores in complex biological organs and tissues, such as melanin, elastin, collagen, keratin, porphyrins and flavins upon excitation by external radiation 8, 9. Optical imaging with non-invasion, real-time, fast feedback and high sensitivity has played a crucial role concerning in vivo bioinformatics visualization 1, 2, 3, 4, 5, 6, 7. Taking advantage of the ATP-responding character, the NIR-II-BPs are able to recognize tumor metastasis with a high tumor-to-normal tissue ratio at 83.4. Their capability of multiplexed imaging is also well displayed. The biocompatible NIR-II-BPs are successfully applied to vessels and lymphatics imaging in mice, which gives ~5 times higher signal-to-noise ratios and ~1.5 times higher spatial resolution than those obtained by NIR-II fluorescence imaging and conventional bioluminescence imaging. To address this challenge, here we present bioluminescence probes (BPs) with emission in the second near infrared (NIR-II) region at 1029 nm by employing bioluminescence resonance energy transfer (BRET) and two-step fluorescence resonance energy transfer (FRET) with a specially designed cyanine dye FD-1029.
However, conventional bioluminescence imaging usually operates in the visible region, which hampers the high-performance in vivo optical imaging due to the strong tissue absorption and scattering. Bioluminescence imaging has been widely used in life sciences and biomedical applications.