The near-infrared-II (NIR-II) in vivo imaging has been proposed as one of the most promising approaches for structural and functional visualization with high resolution and deep penetration1-2. However, the prolonged bioimaging is still a big challenge. Nowadays the most used NIR-II fluorophores are exogenous, suffering from serious limitations, such as non-renewable in living systems, proliferation-related signal dissipation, rapid elimination from the body, and low-biocompatibility3. Endogenous fluorescent proteins (FPs), a kind of biologically rather than chemically synthesized probes, can avoid many of the aforementioned deficiencies. Unfortunately, they are only used for visible and NIR-I imagings4, and their application in the NIR-II region has not been explored.
Here, we report the use of infrared fluorescent proteins (iRFPs) as NIR-II probes to fulfill deeper and prolonged continuous imaging. We find the emission tails of iRFPs extend into the 900-1300 nm region and exhibit high level of brightness (Fig. 1). The in vivo observation indicates that the representative iRFP713 could serve as a fluorescent probe for long-duration NIR-II imaging. To achieve tissue-specific and controlled expression of this probe, iRFP713 is knocked into the mouse genome (Fig. 2). Using partial hepatectomy mouse model, we successfully track the liver regeneration process for a week (Fig. 3). The performance and monitoring efficacy are comparable to that of μCT and superior to that of the clinically approved indocyanine green. Besides, we also effectively monitor the pancreas (Fig. 4), despite its deep location, under both physiological and pathological conditions. This technology permits life-long imaging of the mice.
Fig. 1 Characterization of iRFPs. (a) Normalized fluorescence emission spectra of purified iRFPs. Inset: zoom-in emission spectra in the range of 900-1300 nm. (b) NIR-II fluorescence imaging of purified iRFPs.
Fig. 2 Schematic illustration of generating and NIR-II fluorescence imaging of inducible iRFP713-expressing mice.
Fig. 3 NIR-II in vivo fluorescence imaging of the liver for hepatocyte-specific iRFP713-expressing mice at various time points during liver regeneration. PHX: partial hepatectomy.
Fig. 4 NIR-II in vivo fluorescence imaging of pancreas-specific iRFP713 expressing mice. The control mouse/liver is shown on the left.
In summary, we provide a proof of concept that NIR-II bioimaging of iRFP713 has a long-term monitoring capability with high resolution and deep penetration. Therefore, it may serve as a powerful platform for the imaging of various internal biological processes.
- Feng, Z. et al. Perfecting and extending the near-infrared imaging window. Light Sci. Appl. 10, 197 (2021).
- Qian, Jun, et al. High contrast 3-D optical bioimaging using molecular and nanoprobes optically responsive to IR light. Phys. Rep. 962, 1-107 (2022).
- Jensen, E. C. Use of fluorescent probes: their effect on cell biology and limitations. Anat. Rec. (Hoboken) 295, 2031–2036 (2012).
- Cardarelli, F. Back to the future: genetically encoded fluorescent proteins as inert tracers of the intracellular environment. Int J Mol Sci. 21, 4164 (2020).