Long-term monitoring of intravital biological processes using fluorescent protein-assisted NIR-II imaging

Abstract

High spatial resolution, low background, and deep tissue penetration have made near-infrared II (NIR-II) fluorescence imaging one of the most critical tools for in vivo observation and measurement. However, the relatively short retention time and potential toxicity of synthetic NIR-II fluorophores limit their long-term application. Here, we report the use of infrared fluorescent proteins (iRFPs) as in vitro and in vivo NIR-II probes permitting prolonged continuous imaging (up to 15 months). As a representative example, iRFP713 is knocked into the mouse genome to generate a transgenic model to allow temporal and/or spatial expression control of the probe. To demonstrate its feasibility in a genuine diagnostic context, we adopt two liver regeneration models and successfully track the process for a week. The performance and monitoring efficacy are comparable to those of μCT and superior to those of indocyanine green dye. We are also able to effectively observe the pancreas, despite its deep location, under both physiological and pathological conditions. These results indicate that the iRFP-assisted NIR-II fluorescence system is suitable for monitoring various tissues and in vivo biological processes, providing a powerful noninvasive long-term imaging platform.

Orchestrated Strategies for Developing Fluorophores for NIR‐II Imaging

“…The most critical advances in fluorescence imaging in the last decade include the achievements in ultra-super-high imaging resolution (including spatial sub-diffraction resolution and realtime temporal resolution) and deep tissue imaging, even reach- Reproduced with permission. [53] Copyright 2022, Nature Publishing Group. development of imaging instrumentation, material chemistry, and medicine.…”

“…As mentioned previously, Chen et al selected the brightest fluorescent protein iRFP713 from five fluorescent proteins by comparing their fluorescence intensities. [53] Then, they performed NIR-II fluorescence imaging in mice 10 days after intravenous injection of adeno-associated virus carrying iRFP713, and they could observe clear fluorescence signals with high contrast (Figure 15a), illustrating the potential of iRFP713 for noninvasive NIR-II imaging. Such a biocompatible protein is renewable, because its gene can be genetically encoded into the genome to achieve the amplification of fluorescent signal when the target cells proliferate.…”

“…h) The emission spectra of infrared fluorescent proteins (iRFPs) could extend to the NIR-II window. Reproduced with permission [53]. Copyright 2022, Nature Publishing Group.…”

“…c) NIR-II fluorescence imaging of whole-body iRFP713-expressing mice (iRFP713 flox/flox ; EIIa-Cre) and control littermate mice (iRFP713 flox/flox ) at 1 and 3 weeks after birth. d) NIR-II fluorescence imaging of whole-body iRFP713-expressing mice (iRFP713 flox/flox ; EIIa-Cre) and control littermate mice (iRFP713 flox/flox ) at 8 weeks and 15 months after birth from different views.Reproduced with permission [53]. Copyright 2022, Nature Publishing Group.…”

Aggregation-Induced Emission (AIE), Life and Health

“…The in vivo optical imaging beyond 900 nm has been discovered to possess deeper penetration and higher resolution, compared with imaging in the 360–900 nm range. − The NIR-II (1000–1700 nm) window along with its subwindows including NIR-IIa (1300–1400 nm) and NIR-IIb (1500–1700 nm) window, endow optical molecular imaging with desired penetration depth and spatial-temporal resolution, which has attracted much attention. − Incorporating TPE or bulky modules into the backbone, NIR-II fluorophores can inherit AIE characteristics that not only minimize the quenching effect but also promote the overall fluorescence intensity and the emission tails wavelength moves into the NIR-IIa and NIR-IIb regions. The NIR-II AIE fluorophore, HLZ-BTED based on benzobisthiadiazole (BBTD) and TPE reported by Hong’s group was successfully used for blood vessels, long-term breast tumor, ischemia, the gastrointestinal (GI) tract, and intestinal obstruction NIR-II imaging (Figure A–C) .…”