The NIR second window novel rare earth fluorescent probe is used for in vivo multiplex fluorescence imaging

Fluorescence imaging technology has the characteristics of non-invasive, instant feedback, high sensitivity and high spatial resolution, which makes it irreplaceable in the field of biomedical imaging. The emergence of multiplex fluorescence imaging technology that simultaneously labels multiple analytes with the help of multiple fluorescent probes provides an effective research method for studying complex physiological-pathological mechanisms. However, in practical applications, this technology still has many challenges such as shallow imaging depth, low imaging resolution and signal-to-noise ratio, and the inability to multi-channel dynamic real-time imaging, among which the lack of efficient near-infrared fluorescence probes and instruments capable of real-time multiplex fluorescence imaging are crucial factors hindering the further development of this technology. Therefore, can a series of near-infrared fluorescence-enhanced probes and matching multi-channel real-time imaging devices be developed to solve the above problems?

On June 22, 2023, Beijing time, the journal Nature Nanotechnology published online the scientific research results of Zhang Fan, a professor of the Department of Chemistry of Fudan University, “Fluorescence amplified nanocrystals in the second near-infrared window for in vivo.” real-time dynamic multiplexed imaging”), which provides a new idea for overcoming the above problems. This is also another major achievement achieved by Fudan University through interdisciplinary research.

Yiwei Yang and Ying Chen, 2019 doctoral students in the Department of Chemistry, Fudan University, are the first authors; Professor Zhang Fan and Young Researcher Fan Yong of the Department of Chemistry of Fudan University are the corresponding authors.

Technological progress: NIR fluorescence imaging is gradually applied to real-time dynamic in vivo multiplexing

Fluorescence is a photoluminescent phenomenon in nature. Due to its high sensitivity, instant feedback, and convenient operation, fluorescence imaging has great advantages in clinical medical diagnosis, basic biological exploration and anatomical structure research. With the help of multiplex fluorescence imaging technology in which multiple fluorescent probes label multiple analytes at the same time, researchers can dynamically track the activities of multiple analytes in real time, which is conducive to revealing the complex physio-pathological mechanisms of organisms.

At present, the imaging technology is mainly concentrated in the visible region (400-650 nm) and the near-infrared region (650-900 nm), due to the absorption and scattering of the light in this window by biological tissues, the optical penetration depth and imaging resolution in this window are not ideal. In order to solve this problem, researchers usually use the method of surgical window opening to expose the studied site, hoping to more accurately understand the physiological mechanism of the living in situ microenvironment, but the window inevitably causes damage to the normal physiological environment and brings uncontrollable interference to the detection results. Therefore, how to achieve multiplex fluorescence imaging in deep tissues is a crucial issue that hinders the further development of this technology.

Recent studies have shown that light in the second window of NIR (1000-1700 nm) is subject to less scattering than visible light and NIR zone light and autofluorescence background noise when propagating in biological tissues such as skin, fat and bone. Especially for the sub-imaging window with a wavelength of 1500-1700 nm, the tissue scattering is further reduced, and the autofluorescence background noise of the organism almost disappears, so it is considered to be a biological “transparent” window with great development potential for high-resolution and high-signal-to-noise ratio imaging of living deep tissue. However, the dynamic multiplex in vivo fluorescence imaging research located in this “transparent” imaging window is still not ideal, on the one hand, it is limited by the fluorescent probes available in this imaging window, and only rare earth fluorescence probes based on Er3+ and semiconductor quantum dots with large half-peak width have been reported; On the other hand, there is a lack of devices and technologies that can perform real-time multiplex fluorescence imaging, so real-time dynamic multiplex fluorescence imaging in vivo cannot be achieved.

Research breakthrough: Development of fluorescence-enhanced near-infrared rare earth fluorescence probes and dual-channel fluorescence imaging devices to achieve real-time dynamic multiplex in vivo fluorescence imaging

In view of the above problems, Zhang Fan’s team developed a series of cubic crystal phase rare earth alkali metal fluoride nanofluorescent probes, and built a dual-channel fluorescence imaging device, which realized real-time dynamic multiplex imaging in vivo in the 1500-1700 nm band. In traditional research, rare earth alkali metal fluoride (β-NaREF4) of hexagonal crystal phase has a smaller phonon energy, resulting in a lower probability of non-radiative relaxation, which is generally considered to be more conducive to improving luminous efficiency, so it is widely used as a classical rare earth probe matrix. The Zhang Fan team found that compared with the β-NaREF4 matrix, in the alkali metal fluoride (α-NaREF4) matrix of the cubic crystal phase, the Tm3+ doped rare earth fluorescent probe had nearly 100-fold down-transfer luminescence enhancement at 1632 nm. Raman spectroscopy, variable temperature fluorescence and photon number tests show that phonons with higher α-NaREF4 matrix can effectively promote the electron of Tm3+ from the 3H4 energy level to the 3F4 energy level through non-radiative transition, thereby enhancing the electron distribution of the 3F4 energy level, and the cross-relaxation between the activator ions in the cubic phase matrix and the energy transfer process between the activator ions and the sensitizer ions further lead to the downward transfer luminescence enhancement of Tm3+ at 1632 nm. Based on this fluorescence enhancement mechanism, the Er3+ and Ho3+ doped near-infrared rare earth fluorescence probes were also enhanced to varying degrees of downtransfer luminescence at 1530 nm and 1180 nm. The new TM3+ doped NIR rare earth fluorescence probe provides a new wavelength option for NIR II multiplex fluorescence imaging.

Figure 1: Schematic diagram of the core-shell structure of (a-b) TM3+ doped cubic phase nanoparticles and electron microscopy; (c-d) Tm3+ doped emission spectra of cubic phase and hexagonal crystal phase nanoparticles and luminous intensity histograms at different wavelengths; (e) Cryogenic absorption spectroscopy; (f) Emission spectra of cubic phase nanoparticles doped with Tm3+, Er3+ and Ho3+ and absorption and scattering curves of fat emulsions; (g) Energy transfer mechanism of the YB-TM system; (h) Emission spectra and fluorescence imaging of cubic and hexagonal nanoparticles doped with Er3+ and Ho3+ elements.

For the series of near-infrared second-window fluorescence-enhanced new rare earth fluorescence probes, Zhang Fan’s team further developed a high-spatiotemporal synchronous real-time dynamic multiplex imaging device. Compared with the conventional system of multi-channel imaging by switching optical filters, the imaging device can collect the fluorescence signals of two different channels synchronously in real time, and the simulation experiments of different microsphere motion modified by different fluorescent probes at the same time in vitro also verify that the device can ensure the highly synchronized spatiotemporal imaging of the two channels, laying the foundation for a variety of new near-infrared rare earth fluorescent probes for real-time dynamic multiplex fluorescence imaging in vivo.

Finally, Zhang Fan’s team verified the feasibility of this imaging technique to explore the physiological activity mechanism of deep tissue at the level of fine structure of biological tissues. Firstly, by functionalizing the surface of different near-infrared rare earth fluorescent probes, the differentiation of blood vessels at all levels in the brain vascular network of living mice was realized. The team then used hormone-stimulating mice to simulate the regulation of blood flow by nerves, and the imaging technology was able to achieve real-time dynamic monitoring of the contraction movement of mouse arterial vessels without opening the cranial window, which is expected to provide more accurate information for hemodynamic studies. In order to further explore the potential of this imaging technology for multiplex fluorescence imaging of living deep tissue, the team used the new near-infrared rare earth fluorescent probe developed to specifically label the neutrophils of mice, and realized the monitoring of immune response at the single-cell level through this imaging technology, which can dynamically monitor the chemotaxis, extravasation, activation and other processes of single neutrophils in the subcutaneous inflammatory site and brain injury site in real time. Compared with traditional imaging methods, the new near-infrared rare earth fluorescence probe and dual-channel real-time imaging technology effectively avoid the interference of tissue damage caused by opening the window to the observation results, and provide a new idea for studying cellular immune response at the in vivo level.

Figure 2: (a-b) In vivo dynamic multiplex imaging scheme based on novel near-infrared fluorescent probes realizes real-time dynamic monitoring of vasomotor motility in the mouse brain. (c-f) The in vivo dynamic multiplex imaging scheme constructed based on the novel near-infrared fluorescent probe realizes real-time dynamic monitoring and analysis of neutrophils in the chemotaxis and extravasation process at the subcutaneous inflammatory site. (g-i) A live dynamic multiplex imaging protocol based on a novel near-infrared fluorescent probe enables real-time dynamic imaging of activated neutrophil immune responses at the site of brain injury in stroke mice.

At present, although the preliminary application results of this research have been achieved, it is necessary to further improve the luminous efficiency of the probe and increase the fluorescence emission channel in the future to meet the needs of higher imaging speed, deeper tissue imaging and higher throughput multiplex detection applications in vivo. In addition, improving the functional modification characteristics of fluorescent probes and enhancing compatibility with cutting-edge biological and imaging technologies still need to be followed. However, the many possibilities illuminated by this scientific research will broaden the research horizons in the fields of chemistry and materials science, biomedical photonics, life sciences, biomedical engineering and medical diagnostics.

The research work has been strongly supported by the Department of Chemistry, Fudan University, the State Key Laboratory of Polymer Engineering, the Shanghai Key Laboratory of Molecular Catalysis and Functional Materials, the National Key R&D Project, the National Natural Science Foundation of China, the Shanghai Municipal Science and Technology Commission and other institutions and projects. (Source: Science Network)

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