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.

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 o...

<ol>
	<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+types+of+inorganic/organic+NIR-IIa/IIb+fluorophores:+from+strategic+design+toward+molecular+imaging+and+theranostics.">Versatile types of inorganic/organic NIR-IIa/IIb fluorophores: from strategic design toward molecular imaging and theranostics.</a> Chemical Reviews. 122 (1), 209-268 (2022).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mn-loaded+apolactoferrin+dots+for+in+vivo+MRI+and+NIR-II+cancer+imaging.">Mn-loaded apolactoferrin dots for in vivo MRI and NIR-II cancer imaging.</a> Journal of Materials Chemistry C. 7 (31), 9448-9454 (2019).</li><li>Zhang, F., Tang, B. 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The authors have nothing to disclose.

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 (&#60;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 extr...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anhydrous pyridine</td>
			<td>Perimed&#160;</td>
			<td>110-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous sodium sulfate</td>
			<td>China national medicines Co.,Ltd</td>
			<td>SY006376</td>
			<td></td>
		</tr>
		<tr>
			<td>Black cardboard</td>
			<td>Suzhou Yingrui Optical Technology Co., Ltd</td>
			<td>AO00158</td>
			<td></td>
		</tr>
		<tr>
			<td>Column chromatography</td>
			<td>Energy Chemical</td>
			<td>E080498</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphenylphosphine palladium dichloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2161-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DSPE-PEG2000</td>
			<td>Ponsure</td>
			<td>PS-E1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium&#160;</td>
			<td>Gibco</td>
			<td>8121587</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Biofroxx</td>
			<td>EZ6789D115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>2166090RP</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>GLPBIO</td>
			<td>GC45487-1</td>
			<td></td>
		</tr>
		<tr>
			<td>K2CO3</td>
			<td>Macklin</td>
			<td>P816305-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>N. N &#39;- dimethylformamide</td>
			<td>China national medicines Co.,Ltd</td>
			<td>02-12-1968</td>
			<td></td>
		</tr>
		<tr>
			<td>NIR-II imaging instrument</td>
			<td>Suzhou Yingrui Optical Technology Co., Ltd</td>
			<td>16011109</td>
			<td></td>
		</tr>
		<tr>
			<td>N-sulfenanilide</td>
			<td>Enerry chemical&#160;</td>
			<td>1250030-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>PdCl2(dppf)2CH2Cl2</td>
			<td>TCI&#160;</td>
			<td>B2064-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>China national medicines Co.,Ltd</td>
			<td>M005197</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetratriphenylphosphine palladium</td>
			<td>Immochem</td>
			<td>1021232-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetratriphenylphosphine palladium</td>
			<td>Sigma-Aldrich</td>
			<td>1021232-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Tributyltin chloride</td>
			<td>Immochem</td>
			<td>QH004335</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimethylchlorosilane</td>
			<td>China national medicines Co.,Ltd</td>
			<td>40060560</td>
			<td></td>
		</tr>
	</tbody>
</table>

a bright nir-ii fluorescence probe for vascular and tumor imaging

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.

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)...

Watch this Scientific Journal Video about A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging at JoVE.com

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

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 ...

High-resolution vascular and tumor imaging was realized by the near-infrared to fluorescent nanoprob, providing an accurate and effective method for directing vascular diseases and cancer. In vivo imaging in the NIR-II window, which enables us to look deeply into living subject, produces opportunities for biomedical research and our clinical applications. Prepare for the near-infrared II or NIR-II imaging by placing commercially available black cardboard in the center of the carrier of the imaging device.Then, place the sample on top of the black cardboard, so it is in the center of the carrier. After selecting the appropriate filter, perform the platform height adjustment. Long press the option of platform up on the touchscreen interface of the carrier console control area to move it upwards.And long press the platform down to move it downwards. After synthesizing the NIR-II dye HLY1, prepare the HLY1 dots by nanoprecipitation method using DSPE-PEG-2K as an encapsulation matrix. To do so, place a beaker containing a solution of 10 milligrams of DSPE-PEG-2K in nine milliliters of water for sonication at 25 degrees Celsius.Then, dissolve one milligram of HLY1 in one milliliter of tetrahydrofuran or THF. And slowly add the solution into the DSPE-PEG-2K aqueous solution undergoing sonication. Subsequently, remove THF from the mixture by dialysis.After concentrating the above solution centrifugally with ultrafiltration at 7, 100 G for 10 minutes, place it in a four degree Celsius refrigerator for future use. For in vivo imaging, inject the HLY1 dots intravenously into the anesthetized mice. And three minutes later, proceed to perform NIR-II fluorescence imaging of the blood vessels of the whole body of the mice using an optical NIR-II imaging system.Focus further on the mouse's head to collect brain vascular imaging. Set the instrument parameters of the optical NIR-II imaging system at 90 milliwatts per square centimeter and 808 nanometer laser. Collect the images five minutes after the injection of HLY1 dots in mice and process the data using ImageJ software.The fluorescence intensity of HLY1 in the 90%THF solution containing 90%water was five times that in the THF solution, indicating a prominent aggregation-induced emission or AIE feature of HLY1. The HLY1 dots emitted strong fluorescent signals under a 1, 500 nanometer low pass or LP filter establishing its suitability for NIR-IIb imaging. The maximum absorption and emission wavelength of HLY1 dots were 740 nanometers and 1, 040 nanometers, respectively.Additionally, the hydrodynamic size of HLY1 dots was determined to be 145 nanometers by dynamic light scattering. In mice administered with HLY1 dots, the cerebral vessels and the micro vessels in the hind limb were identified clearly by NIR-II imaging under a 1, 500 nanometer LP filter. Further, the four T1 tumor of the tumor-bearing mice was also clearly visible by NIR-II imaging, indicating the enhanced permeability and retention or EPR effect of HLY1 dots.All these results suggested that HLY1 dots are a bright NIR-II fluorescence probe applicable for vascular and tumor imaging. Preparation of the water-suspendable nanoprobe under the in vivo and NIR-II fluorescence imaging are the most important part of the procedure.

a-bright-nir-ii-fluorescence-probe-for-vascular-and-tumor-imaging

Research

JoVE Journal

Medicine

1.5K Views.  Tibet University. High-resolution vascular and tumor imaging was realized by the near-infrared to fluorescent nanoprob, providing an accurate and effective method for directing vascular diseases and cancer. In vivo imaging in the NIR-II window, which enables us to look deeply into living subject, produces opportunities for biomedical research and our clinical applications. Prepare for the near-infrared II or NIR-II imaging by placing commercially available black cardboard in the center of the carrier of the im...