Introduction Fluorophores emitting in the second near-infrared window (NIR-II, 900-1700 nm)[1] have exhibited significant improvements in detection depth and resolution owing to low tissue autofluorescence and up to a 1000-fold reduction in scattering losses compared to those for visible (400-780 nm)[2] and NIR-I regions (780-900 nm)[3]. Especially, the range of 1500-1700 nm is denoted as the “NIR-IIb” region, which provides the lowest photon scattering in the NIR-II region, thus allowing for the most desirable imaging clarity and deep penetration using existing InGaAs detectors [4]. Unfortunately, NIR-IIb fluorescent probes with sufficient brightness are scarce. Herein, we present an efficient NIR-IIb imaging agent NaErF4:0.5%Tm3+@NaLuF4 with a shell thickness of 4.30 nm. The influence of the types of epitaxial heterogeneous shells, the doping effect and optimal doping concentration of Tm3+ ions, as well as the critical shell thickness for obtaining the surface quenching-assisted downshifting were systematically investigated to acquire efficient 1525 nm emission of Er3+ ions under 800 nm excitation. The quantum yield in 1500-1700 nm region reached to 13.92%, enabling high-resolution through-skull microscopic cerebrovascular imaging and large-depth in vivo gastrointestinal tract imaging with extremely low excitation powder density of 35mW/cm2. Method The 793 nm laser beam emitted by the semiconductor laser (Suzhou Rugkuta Optoelectronics Co., Ltd., China) was collimated and expanded through the lens so that the sample can be uniformly illuminated. The emitting fluorescent signal was collected onto the InGaAs camera (Tekwin, China) through a prime lens (focal length: 50 mm, Edmund Optics). A 1450 nm long-pass filter (Thorlabs) was placed in front of the detector to extract the pure NIR-IIb signals. After anesthesia, the mice were placed on the imaging platform. The PEGylated β-NaErF4:0.5%Tm@4.30nm NaLuF4 nanoparticles (200 μL, 5 mg/mL) was injected into the tail vein, then the laser was turned on immediately and the image was recorded at different time points.The β-NaErF4:0.5%Tm@4.30nm NaLuF4 nanoparticles (200 μL, 5 mg/mL) were perfused into the stomach of nude mice. The images of the mice were recorded using the above-mentioned NIR-IIb fluorescence macroscopic imaging system. Results and Conclusions The brightest β-NaErF4:0.5%Tm@4.30nm NaLuF4 nanoparticels (NPs) were used for the in vivo bio-imaging. Firstly, 200 μL NPs (PEGylated β-NaErF4:0.5%Tm@4.30nm NaLuF4, 5mg/mL) were intravenously injected into the mouse for the whole body imaging (Fig.1), which verified the feasibility of the probe for NIR-IIb in vivo imaging. Under the excitation of a 793 nm laser (~35 mW/cm2), a bright, clear vascular network in the nearly “transparent” body was presented. Within 5 min post-injection, it could be seen that the liver has been gradually brighter, for which the NPs were captured by the liver (Fig.1B). Thanks to the low autofluorescence and suppressed tissue scattering in NIR-IIb region, a clear outline of liver was distinguished. It is worth noticing that, owing to the excellent luminescence properties of the probe, the excitation power density at the imaging plane could be as low as ~35 mW/cm2, which is much smaller than that in previously reported work.[5] The deep-tissue penetration in the NIR-IIb region was well validated in Fig. 2. After anesthesia, a mouse with a cranial window and a mouse with intact skull were injected with PEGylated β-NaErF4:0.5%Tm@4.30nm NaLuF4 NPs (200 μL, 100 mg/mL) respectively and fixed in the imaging system for microscopic cerebrovascular imaging. The 793 nm laser beam was expanded, reflected, and uniformly illuminated in the region of interest. The NIR-IIb fluorescence signals emitted by the NPs in the blood vessel were collected by the objective lens and focused by the tube lens onto the InGaAs detector (Tekwin, China). In order to visually show the structure of the blood vessels in the mouse brain, we stitched several images recorded by a scan lens (LSM03, Thorlabs) into an image with a large field of view. Compared to open-skull brain imaging (Fig.2A), NIR-IIb through-skull imaging (Fig.2B) also provided fairly rich details, and a small blood vessel with the diameter of 18.609 μm was easily identified.
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Open Access
Jul 1, 2021
Minmin Zhao + 4
