Name: A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging (Video) | JoVE Journal
Uploaded: 2023-03-17T14:55:00.000+00:00
Duration: 5 min 51 s
Description: Learn NIR-II fluorescence imaging for vascular and tumor studies in live mice using HLY1 nanoparticles. This protocol demonstrates high-resolution visualization of cerebral vessels and 4T1 tumors.

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5 minutes

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 of age were used for the present study.
1. NIR-II imaging preparation
<ol>
	<li>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.</li>
	<li>Select a suitable filter based on the wavelength of the NIR-II probe. Press long (&#62;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.</li>
	<li>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.</li>
	<li>Adjust the platform height to &#34;0 mm&#34; (height adjustment) and use automatic focusing to make the NIR-II image clear.</li>
</ol>
2. Synthesis of NIR-II dye (HLY1)
<ol>
	<li>Weigh the raw materials required for the synthesis experiment. Ensure they do not deteriorate.</li>
	<li>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&#160;(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 &#176;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.</li>
	<li>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).</li>
	<li>Place the dye HLY1 under the protection of nitrogen in the refrigerator for later use. This can be stored for up to 6 months.</li>
</ol>
3. Preparation of water-suspensible nanoprobe
<ol>
	<li>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).</li>
	<li>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 &#176;C. Subsequently, remove THF from the mixture by dialysis20.</li>
	<li>Concentrate the above solution centrifugally with ultrafiltration18 (7100 x g for 10 min) and then place it in a 4 &#176;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 &#176;C and used as soon as possible.</li>
</ol>
4. Construction of tumor-bearing mice
<ol>
	<li>Culture 4T1 mouse breast cancer cells (4T1) in Dulbecco&#39;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 &#176;C.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Then, using an insulin injection needle, inject the 4T1 cell mixture into the mice through subcutaneous injection (100 &#181;L). 
	&#8203;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.</li>
</ol>
5. In vivo NIR-II fluorescence imaging
<ol>
	<li>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.</li>
	<li>Take a solution of HLY1 dots (0.8 mg/mL, 200 &#181;L). 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&#39;s head to collect brain vascular imaging. 
	NOTE: Use clean experimental gloves during imaging, which will help to obtain clean NIR-II images.</li>
	<li>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).</li>
	<li>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.</li>
</ol>

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</ol>

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><tbody><tr><td>Anhydrous pyridine</td><td>Perimed&amp;nbsp;</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&#039;s modified eagle medium&amp;nbsp;</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>K&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Macklin</td><td>P816305-5g</td><td></td></tr><tr><td>N. N &#039;- 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&amp;nbsp;</td><td>1250030-5g</td><td></td></tr><tr><td>PdCl&lt;sub&gt;2&lt;/sub&gt;(dppf)&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;</td><td>TCI&amp;nbsp;</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

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)...

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 ...

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

Research

JoVE Journal

Medicine

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo.  The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes.   The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy...

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

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Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Learn NIR-II fluorescence imaging for vascular and tumor studies in live mice using HLY1 nanoparticles. This protocol demonstrates high-resolution visualization of cerebral vessels and 4T1 tumors.

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

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