The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.
As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.
Fluorescence imaging is the commonly used molecular imaging tool in basic research, and is also often used to guide surgical tumor resection in clinics1. The essential principle of fluorescence imaging is to employ a camera to receive fluorescence emitted by a laser after the irradiation of samples (tissues, organs, etc.)2. The process is completed within a few milliseconds3. The fluorescence imaging wavelengths can be divided into ultraviolet (200-400 nm), visible region (400-700 nm), near-infrared I (NIR-I, 700-900 nm), and near-infrared II (NIR-II, 1000-1700 nm)4,5,6. Because the endogenous molecules such as hemoglobin, melanin, deoxyhemoglobin, and bilirubin in biological tissues have strong absorption and a scattering effect on the light in visible regions, the penetration and sensitivity of light are greatly reduced, and the fluorescence imaging in visible light wavelengths is adversely affected7,8,9.
NIR-II fluorescence imaging has low photon absorption and scattering, high imaging speed, and high image contrast (or sensitivity)10,11. As the fluorescence wavelength increases, the absorption and scattering of fluorescence in biological tissues decrease gradually, and the auto-fluorescence in the NIR-II region is extremely low12. Thus, the NIR-II window significantly increases the penetration depth of tissues and obtains a higher resolution and signal-to-noise ratio13,14,15. The NIR-II window can be further subdivided into the NIR-IIa (1300-1400 nm) and NIR-lIb (1500-1700 nm) windows16. To date, several milestone NIR-II materials have been reported, including inorganic material single-walled carbon nanotubes, rare earth nanoparticles, quantum dots, and organic material semiconductor polymer nanoparticles, small-molecule dyes, aggregation-induced luminescent materials, etc.1,17,18,19,20,21,22. Inorganic nanomaterials are easily accumulated in the liver, spleen, etc., and have potential long-term biotoxicity23. Organic small-molecule fluorophore has the advantages of rapid metabolism, low toxicity, easy modification, and a clear structure, which is the most promising probe for clinical use24.
The NIR-II optics imaging system is also a critical component of fluorescence bioimaging because it can efficaciously collect NIR-II fluorescence signals from the NIR-II probe, thus rendering precise functional, anatomical, and molecular images25,26. The NIR-II imaging system mainly comprises shortwave infrared cameras, long-pass (LP) filters, lasers, and computer processors. In vivo NIR-II fluorescent imaging is considered one of the most feasible imaging approaches for elucidating the mechanisms of diseases and the nature of life27,28,29. NIR-II imaging technology has been widely used in biomedical fields such as cancer cell detection, dynamic imaging, in vivo targeted tracing, and targeted therapy, especially in oncology research30,31. However, considering the high technical requirements of NIR-II imaging technology on imaging probes and instruments, it also puzzles and restricts the practical use of researchers in different fields. Therefore, the preparation of NIR-II imaging probes and the applications of NIR-II imaging are introduced in detail in this article.
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Animal experiments for NIR-II imaging studies were conducted at the Animal Experiment Center of Wuhan University, which has been awarded the International Association for Experimental Animal Care (AALAC). All animal studies were conducted following the China Animal Welfare Commission Guidelines for the Care and Use of Experimental Animals and approved by the Animal Care and Use Committee (IACUC) of the Animal Experimental Center of Wuhan University.
Female BALB/c nude mice (~20 g) at 6 weeks of age were used for the present study.
1. NIR-II imaging preparation
- Place commercially available black cardboard (see Table of Materials) in the center of the carrier. Then, place the sample on top of the black cardboard, so that the sample is in the center of the carrier (a stage located in the imaging device).
NOTE: Compared with white cardboard, black cardboard has less background interference during NIR-II imaging.
- Select a suitable filter based on the wavelength of the NIR-II probe. Press long (>2 s) to control the box area (such as 900 LP) corresponding to the filter model in the screen interface when the system moves the filter into the optical imaging path.
- Long press platform up on the touch screen interface of the carrier console control area so that the carrier consoles up; long press platform down so the carrier consoles down.
- Adjust the platform height to "0 mm" (height adjustment) and use automatic focusing to make the NIR-II image clear.
2. Synthesis of NIR-II dye (HLY1)
- Weigh the raw materials required for the synthesis experiment. Ensure they do not deteriorate.
- Add Compound 1 (200 mg, 0.18 mmol), PdCl2(dppf)2CH2Cl2 (28 mg, 0.04 mmol), N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-2-amine (170 mg, 0.4 mmol), and K2CO3 (46 mg, 0.34 mmol) to tetrahydrofuran (THF) solution in a 25 mLround-bottom flask. Stir the mixture for 4 h at 75 °C under N2 atmosphere (Figure 1A).
NOTE: For the synthesis procedure of Compound 1 and N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-2-amine, refer to Li et al21. The chemical structures are shown in Figure 1A.
- After cooling to ambient temperature, quench the reaction with distilled (DI) water (80 mL), and extract the mixture with DCM (dichloromethane)/H2O (30 mL) (three times). Purify the crude product by column chromatography16 (petroleum ether:DCM = 10:1) to make HLY1 a green solid (78 mg, 30% yield).
- Place the dye HLY1 under the protection of nitrogen in the refrigerator for later use. This can be stored for up to 6 months.
3. Preparation of water-suspensible nanoprobe
- Weigh HLY1 (1 mg) and amphipathic encapsulation materials, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2k (DSPE-PEG2k, 10 mg; see Table of Materials).
- Prepare HLY1 dots20 by employing DSPE-PEG2k as an encapsulation matrix (nanoprecipitation method12) (Figure 1C). Dissolve HLY1 in THF (1 mL) and slowly add into a beaker containing DSPE-PEG2k aqueous solution (9 mL) with sonication at 25 °C. Subsequently, remove THF from the mixture by dialysis20.
- Concentrate the above solution centrifugally with ultrafiltration18 (7100 x g for 10 min) and then place it in a 4 °C refrigerator for future use. This can be stored for up to 1 month.
NOTE: The nanoprobe aqueous solution loaded by DSPE-PEG2k should be stored above 0 °C and used as soon as possible.
4. Construction of tumor-bearing mice
- Culture 4T1 mouse breast cancer cells (4T1) in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (see Table of Materials), and maintain in a humidified incubator with 5% CO2 at 37 °C.
- For the NIR-II fluorescent imaging experiment, culture 4T1 cells (5 x 107) for 24 h, digest with trypsin (1 mL), and wash twice with serum-free DMEM (4 mL).
- Anesthetize the mice by treating with isoflurane (2%). Confirm adequate anesthetization by stimulating the toes or the soles of the feet of the mice, and observe whether the mice respond. If there is no response, it means that the anesthesia is sufficient32.
- Then, using an insulin injection needle, inject the 4T1 cell mixture into the mice through subcutaneous injection (100 µL).
NOTE: NIR-II imaging studies were performed ~2 weeks after inoculation, when the tumor had grown to a volume of ~100 mm3. Before NIR-II tumor imaging, please confirm the tumor size. The tumor size was estimated by an electronic vernier caliper for the present study11.
5. In vivo NIR-II fluorescence imaging
- Anesthetize the mice by treating with isoflurane (2%) and perform NIR-II imaging of the whole body of the mice using an optical NIR-II imaging system (see Table of Materials).
NOTE: Pay attention to the dosage of anesthetic to avoid mice death. Generally, anesthesia lasts for 5-10 min. Stimulate the toes or the soles of the feet of the mice, and observe whether the mice respond. If there is no response, it means that the anesthesia is sufficient.
- Take a solution of HLY1 dots (0.8 mg/mL, 200 µL). Inject the HLY1 dots intravenously into the anesthetized mice, and 3 min later, perform NIR-II fluorescence imaging of the blood vessels of the whole body of mice using an NIR-II imaging system. Focus further on the mouse's head to collect brain vascular imaging.
NOTE: Use clean experimental gloves during imaging, which will help to obtain clean NIR-II images.
- Collect the images 5 min after the injection of HLY1 dots in mice, and process the data using ImageJ software. The instrument parameters of the optical NIR-II imaging system are 90 mW/cm2 (808 nm laser).
- On completion of the experiment, euthanize the animals following institutionally approved protocols.
NOTE: For the present study, the animals were euthanized by exposing them to excess isoflurane32.
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The fluorescent intensity and brightness of water-suspensible HLY1 dots were determined by an NIR-II imaging instrument. The fluorescent intensity of HLY1 in the 90% fwTHF/H2O mixture was five times that in the THF solution, which indicated a prominent AIE feature of HLY1 (Figure 1B). Moreover, HLY1 dots emitted strong fluorescent signals under a 1,500 nm LP filter, showing that HLY1 dots can be used for NIR-IIb imaging (Figure 1D). The maximum absorption and maximum emission wavelength of HLY1 dots were 740 nm and 1,040 nm, respectively (Figure 2A). Moreover, the hydrodynamic size of HLY1 dots was determined to be 145 nm by dynamic light scattering (DLS) (Figure 2B). HLY1 dots (0.2 mL, 0.8 mg/mL) were administered into normal Balb/c mice via tail vein injection for vascular imaging (Supplementary Figure 1). The micro-vessels in the hindlimb were identified clearly under a 1,500 nm LP filter (Figure 3B). In addition, the cerebral vessels were also clearly identified under a 1,500 nm LP filter (Figure 3A). The NIR-II imaging performance of the HLY1 dots in 4T1 tumor-bearing mice was also evaluated through the NIR-II imaging system. HLY1 dots (0.2 mL, 0.8 mg/mL) were intravenously injected into 4T1 mice through the tail vein. The 4T1 tumor of the tumor-bearing mice was clearly visible by NIR-II imaging (Figure 3C), indicating the EPR effect of HLY1 dots. All these results suggest that HLY1 dots are a bright NIR-II fluorescence probe, which is applicable for vascular and tumor imaging.
Figure 1: Synthesis of dye molecules and preparation of water-suspensible probes. (A) The synthetic path of HLY1 (a: Pd(dppf)Cl2 CH2Cl2, K2CO3, 75 °C). (B) The NIR-II images of HLY1 in THF and 90% fw THF/H2O (1,000 nm LP, 2 ms). (C) A schematic diagram of the preparation of HLY1 dots. (D) The NIR-IIb fluorescent intensity of HLY1 dots in aqueous solution (1,500 nm LP, 200 ms). Please click here to view a larger version of this figure.
Figure 2: The optical properties and hydrodynamic size of HLY1 dots. (A) The absorption and emission spectra of HLY1 dots in aqueous solution. (B) The DLS of HLY1 dots. Please click here to view a larger version of this figure.
Figure 3: NIR-II fluorescence imaging using HLY1 dots. (A) Brain vascular imaging in mice (1,500 nm LP, 300 ms exposure time). Scale bar: 2 cm. (B) Whole body vascular imaging in mice (1,500 nm LP, 300 ms). (C) 4T1 tumor imaging (1,250 nm LP, 30 ms). Scale bar: 1 cm. Please click here to view a larger version of this figure.
Supplementary Figure 1: NIR-II imaging setup. (A) Schematic diagram of injection of HLY1 dots into mice. (B) The photograph of the NIR-II imaging device. Please click here to download this File.
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NIR-I fluorescent imaging can be used to some extent for tumor and vascular imaging, but due to the limited maximum emission wavelength of NIR-I fluorophores (<900 nm), it results in poor tissue penetration and tumor signal background ratio33,34. Poor and low imaging resolution may cause a deviation between the outcome of the imaging feedback treatment and the actual therapeutic effect. In addition, most NIR-I fluorophores have poor optical stability and extremely fast metabolism, resulting in instability in the imaging process. Because of the low tissue penetration and instability of NIR-I fluorophores, the application in tumor and vascular imaging is greatly limited35. Compared with NIR-I light, NIR-II fluorescence imaging has the advantages of significantly reduced photon scattering and absorption, lower tissue auto-fluorescence, stronger body tissue penetration, and better imaging spacetime resolution36.
This article describes a bright AIE dye based on a D-A-D skeleton, which has excellent stability. An effective nanoprecipitation method was utilized to prepare a nanoprobe for multi-purpose bioimaging, including vascular diseases and tumor imaging. The high quantum yield in the aqueous solution is due to the luminescent properties induced by aggregation, which can achieve high-definition NIR-II imaging with low dose and high biosecurity. The brightness of the NIR-II probe and the water solubility determine the quality of the imaging. Additionally, when injecting a probe into a mouse, it is necessary to avoid leaking the probe into the mouse's tail, which affects the accuracy of the imaging results. The current method of administration is only limited to intravenous injection, and cannot use multiple injection methods, which is a limitation of the current method. In addition, the NIR-II nanoprobe of this method can only be accumulated to the target by passive targeting, and cannot identify specific targets by active targeting.
In the process of implementing NIR-II imaging, the operation of the NIR-II device is also important for the acquisition of images. To obtain high-resolution vascular imaging, the InGaAs camera needs to be focused on the mouse and positioned close to the mouse, making it easy to observe the tiny blood vessels. For tumor imaging, probes need to be effectively accumulated into the tumor, and NIR-II fluorescence should be emitted by the probes accumulated in the tumor, effectively distinguishing the boundary between the tumor and the surrounding tissue. Because of the high sensitivity of NIR-II fluorescence imaging, images can be observed dynamically during imaging, which is lacking in many other imaging techniques.
In this study, the preparation of a fluorescent probe is introduced. At the same time, high-resolution vascular and tumor imaging is realized by an NIR-II fluorescent nanoprobe, which provides an accurate and effective method for the detection of vascular diseases and cancer.
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The authors have nothing to disclose.
This work was partially supported by grants from NSFC (82273796, 82111530209), Special Funds for Guiding Local Science and Technology Development of Central Government (XZ202202YD0021C, XZ202102YD0033C, XZ202001YD0028C), Hubei Province Scientific and Technical Innovation Key Project (2020BAB058), the Fundamental Research Funds for the Central Universities, and the Tibet Autonomous Region COVID-19 Prevention and Control Programs for Science and Technology Development.
|Anhydrous sodium sulfate||China national medicines Co.,Ltd||SY006376|
|Black cardboard||Suzhou Yingrui Optical Technology Co., Ltd||AO00158|
|Column chromatography||Energy Chemical||E080498|
|Diphenylphosphine palladium dichloride||Sigma-Aldrich||B2161-1g|
|Dulbecco's modified eagle medium||Gibco||8121587|
|Fetal bovine serum||Gibco||2166090RP|
|N. N '- dimethylformamide||China national medicines Co.,Ltd||02-12-1968|
|NIR-II imaging instrument||Suzhou Yingrui Optical Technology Co., Ltd||16011109|
|Tetrahydrofuran||China national medicines Co.,Ltd||M005197|
|Trimethylchlorosilane||China national medicines Co.,Ltd||40060560|
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