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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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

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 ...

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

Research

JoVE Journal

Medicine

1.7K Views.  Tibet University. Hoge resolutie vasculaire en tumor beeldvorming werd gerealiseerd door de nabij-infrarode tot fluorescerende nanoprob, die een nauwkeurige en effectieve methode biedt voor het richten van vaatziekten en kanker. In vivo beeldvorming in het NIR-II-venster, dat ons in staat stelt om diep in levende onderwerpen te kijken, biedt kansen voor biomedisch onderzoek en onze klinische toepassingen. Bereid u voor op de nabij-infrarood II- of NIR-II-beeldvorming door in de handel verkrijgbaar zwart karton...

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

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

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

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

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 that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

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

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

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.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Cancer Research

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengineering

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

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Immunology and Infection

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

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.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

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

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

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

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.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

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

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.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

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

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

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.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

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

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

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.

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

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