Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

Multimodality imaging of endothelial progenitor cells with a novel multifunctional probe featuring positive magnetic resonance contrast and near-infrared fluorescence

The multimodal strategy incorporating T1-weighted magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence imaging can complement their strengths to provide images with high sensitivity and spatial resolution for noninvasively and dynamically monitoring endothelial progenitor cells (EPCs) in potential EPC-dominated therapies. Here we report the development of a protein-based imaging probe, bCD-PLL-Cy5.5 Conjugate 1, in which the bacterial cytosine deaminase (bCD) protein was modified with poly-l-lysine (PLL) that is labeled with imaging reporters, including T1-weighted MRI contrast chelator and NIR fluorophore. Conjugate 1 showed low cytotoxicity in EPCs isolated from the rabbit peripheral blood. The normalized cell viability was maintained above 90% after incubation for 1 to 5 days. Fluorescence microscopy of live cells indicated rapid cellular uptake of Conjugate 1 into EPCs in 15 minutes, and flow cytometry studies demonstrated the time-dependent internalization of Conjugate 1 with maximum uptake 48 hours after the treatment. MRI of phantoms demonstrated significant reduction of the T1 value of the EPC pellet that was pretreated with 2 μM of Conjugate 1 for 24 hours. Our preliminary data suggest that as a multimodal imaging contrast medium, Conjugate 1 offers a promising imaging probe for tracking the delivery and therapeutic response of EPCs in vivo.     

A liposomal system for contrast-enhanced magnetic resonance imaging of molecular targets

Pegylated paramagnetic and fluorescent immunoliposomes were designed to enable the parallel detection of the induced expression of molecular markers on endothelial cells with magnetic resonance imaging (MRI) and fluorescence microscopy. MRI is capable of three-dimensional noninvasive imaging of opaque tissues at near cellular resolution, while fluorescence microscopy can be used to investigate processes at the subcellular level. As a model for the expression of a molecular marker, human umbilical vein endothelial cells (HUVEC) were treated with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) to upregulate the expression of the adhesion molecule E-selectin/CD62E. E-selectin-expressing HUVEC were incubated with pegylated paramagnetic fluorescently labeled liposomes carrying anti-E-selectin monoclonal antibody as a targeting ligand. Both MRI and fluorescence microscopy revealed the specific association of the liposomal MR contrast agent with stimulated HUVEC. This study suggests that this newly developed system may serve as a useful diagnostic tool to investigate pathological processes in vivo with MRI.     

Hyperbranched polyglycerols as trimodal imaging agents: design, biocompatibility, and tumor uptake

Combining various imaging modalities often leads to complementary information and synergistic advantages. A trimodal long-circulating imaging agent tagged with radioactive, magnetic resonance, and fluorescence markers is able to combine the high sensitivity of SPECT with the high resolution of MRI over hours and days. The fluorescence marker helps to confirm the in vivo imaging information at the microscopic level, in the context of the tumor microenvironment. To make a trimodal long-circulating probe, high-molecular-weight hyperbranched polyglycerols (HPG) were modified with a suitable ligand for (111)In radiolabeling and Gd coordination, and additionally tagged with a fluorescent dye. The resulting radiopharmaceutical and contrast agent was nontoxic and hemocompatible. Measured radioactively, its total tumor uptake increased from 2.6% at 24 h to 7.3% at 72 h, which is twice the increase expected due to tumor growth in this time period. Both in vivo MRI and subsequent histological analyses of the same tumors confirmed maximum HPG accumulation at 3 days post injection. Furthermore, Gd-derivatized HPG has an excellent contrast enhancement on T1-weighted MRI at 10× lower molar concentrations than commercially available Galbumin. HPG derivatized with gadolinium, radioactivity, and fluorescence are thus long-circulating macromolecules with great potential for imaging of healthy and leaky blood vessels using overlapping multimodal approaches and for the passive targeting of tumors.     

Magneto/optical annexin V, a multimodal protein

Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC(50) of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO-Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.     

Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

WGA-QD probe-based AFM detects WGA-binding sites on cell surface and WGA-induced rigidity alternation

A strategy involving the conjugation of fluorescent quantum dot (QD) with wheat germ agglutinin (WGA) acting as fluorescent and topographic probes prior to cell surface staining is developed for fluorescence microscopy and atomic force microscopy (AFM). This strategy provided at least two advantages: (a) an amplified fluorescence of WGA-QD aggregates, strongly resistant to photobleaching, ensures repeated/real-time observations of the probe-labeled cells by fluorescence microscopy; (b) the enlarged size of WGA-QD probe makes it possible for labeled WGA to be distinguished from other membrane proteins by AFM. Here, the random distribution of WGA-binding sites on non-crosslinked cells and the uneven or polarized reorganization due to WGA-induced crosslinking on cell surfaces were studied using AFM-detectable WGA-QD probe. Moreover, we developed a method to rapidly detect the WGA-induced rigidity alternation of the whole cells, which is efficient and has the potentiality of being developed to a useful tool in clinical diagnosis.     

Dual‐function fluorescent probe for cancer imaging and therapy

To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual‐function quantum dot (QD) fluorescent probes with dual‐targeting action and a therapeutic effect are rare. Here, a dual‐targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin–glycyrrhetinic acid conjugate and arginine‐glycine‐aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as‐prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Applications of fluorescent semiconductor nanocrystals in microscopy and cytometry

Quantum dots (QD) nanocrystals consisting of CdSe core with ZnS shell are a novel class of fluorophores with tremendous potential in microscopy and cytometry techniques. The unique optical features of Qdots, namely, high photostability and extinction coefficient, wide absorption and narrow emission spectra, and large Stokes shift make them desirable fluorescent tags for diverse biomedical applications. Applications of this novel technology in microscopy and cytometry produce reliable multicolor specimens due to increased photostability, ability for multiplexing and narrow emission spectra of nanocrystals. QD conjugates are available on the market and could be prepared in the laboratory. This paper describes the application of QD-conjugates for immunophenotyping and FISH assessment of cells and tissues, and the requirements for microscope and flow cytometer reengineering for successful use of QD in multiplex fluorescent format. Despite the considerable progress, important methodological issues still need to be solved in terms of QD nanocrystals' size, heterogeneity, functionalization and stability of their conjugates. We discuss practical approaches and challenges that need to be addressed to make QD immunostaining a standard method in biology.     

Distribution of injected MRI contrast agents in mouse livers studied by confocal and SIMS microscopy

OBJECTIVE: To localize magnetic resonance imaging (MRI) contrast agents injected intravenously into mouse livers. STUDY DESIGN: Parallel studies were performed on fluorescent europium and nonfluorescent, paramagnetic gadolinium and on a product combining nanoparticles of Fe and Texas Red to obtain combined information on the distribution of these molecules inside the liver. The distribution of different superparamagnetic iron oxides was also studied because the size of these new compounds is not always convenientfor microcirculation studies. RESULTS: Europium and Texas Red can be detected by confocal microscopy. Europium, iron and gadolinium can be detected by secondary ion mass spectrometry (SIMS) microscopy. Studies confirmed the complementarity of both microscopies. They also confirmed the possibility of using europium as a model of gadolinium to analyze thefate of MRI contrast agents. CONCLUSION: The methodology can be used on mice injected intravenously and analyzed by confocal and SIMS microscopy to localize MRI contrast agents inside cellular and tissue specimens of mice.     

EPR Oximetry Sensor—Developing a TAM Derivative for In Vivo Studies

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1?mM PBS, pH?=?7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH?=?7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250?mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.     

HER2 targeted molecular MR imaging using a de novo designed protein contrast agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.     

Contrast agents for MRI: 30+ years and where are we going?

Thirty years ago, Schering filed the first patent application for a contrast agent for magnetic resonance imaging (MRI) covering the forefather of the gadolinium contrast agents and still the most widely used gadolinium probe: gadolinium(III) diethylenetriaminepentaacetate (Magnevist). To date, 11 contrast agents have been approved by the US Food and Drug Administration for intravenous use. Coordination chemists have done a great deal to move the field forward. Our understanding of lanthanide chemistry now makes possible the design of complexes with long rotational correlation times, fast or slow water-exchange rates, high thermodynamic stabilities, and kinetic inertness, leading to sensitive and nontoxic contrast agents. Chemists did not stop there. The last few decades has seen the development of novel classes of probes that yield contrast through different mechanisms, such as paramagnetic chemical exchange saturation transfer agents. Thirty years since the first patent, chemists are still leading the way. The development of high-sensitivity contrast agents for high magnetic fields, safe probes for patients with kidney disorders, and multimodal, targeted, and responsive agents demonstrates that the field of contrast agents for MRI still has much to offer.     

转铁蛋白导向脂质体核磁造影剂纳米粒子（TfNIR-LipNBD-Magnevist）——一种肿瘤靶向磁共振造影剂

造影剂辅助的核磁共振成像是目前肿瘤诊断的最吁方法之一。但是由于核磁共振成像内在的低灵敏性以及造影剂的非特异性,导致肿瘤早期诊断较为困难。文章将一种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断。这种新的肿瘤靶向核磁造影剂纳米粒子由配体转铁蛋白（Tf）、纳米水平的正电脂质体（Lip）载体和临床常用的造影剂Magnevist（Tf^NIR-Lip^NBD-Magnevist）三部分构成。另外转铁蛋白和脂质体粒子上,亦标记了荧光物质用于确定转铁蛋白一脂质体一造影剂纳米粒子的靶向性,以及肿瘤的光学影像诊断。在体外实验中,利用激光共聚焦显微镜和光学影像证明了靶向纳米粒子介导的细胞内吞和特异性结合。在裸鼠肿瘤模型中,造影剂纳米粒子Tf^NIR-Lip^NBD-Magnevist经尾静脉注入后,显著增强了肿瘤内信号与周围组织的对比度。由造影剂纳米粒子介导的肿瘤内信号显著强于单独Magnevist辅助的肿瘤内信号。同时,利用光学影像方法,在肿瘤内检测到特异的荧光信号。其结果进一步支持了转铁蛋白一脂质体一造影利（Tf^NIR-Lip^NBD-Magnevist）纳米粒子的靶向性和肿瘤影像诊断的有效性。     

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.     

Biodistribution study of nanometric hybrid gadolinium oxide particles as a multimodal SPECT/MR/optical imaging and theragnostic agent

Nanometric hybrid gadolinium oxide particles (Gado-6Si-NP) for diagnostic and therapeutic applications (mean diameter 3-4 nm) were obtained by encapsulating Gd(2)O(3) cores within a polysiloxane shell, which carries organic fluorophore (Cy 5) and is derivatized by a hydrophilic carboxylic layer. As residency time in the living body and methods of waste elimination are crucial to defining a good nanoparticle candidate and moving forward with steps for validation, this study was aimed at evaluating the biodistribution of these multimodal Gado-6Si-NP in rodents. Gado-6Si-NP were imaged following intravenous injection in control Wistar rats and mice using MRI (7 T), optical fluorescent imaging, and SPECT. A clear correlation was observed among MRI, optical imaging, and SPECT regarding the renal elimination. Quantitative biodistribution using gamma-counting of each sampled organ confirmed that these nanoparticles circulated freely in the blood pool and were rapidly cleared by renal excretion without accumulation in liver and RES uptake. These results demonstrate that Gado-6Si-NP display optimal biodistribution properties, enabling them to be developed as multimodal agents for in vivo imaging and theragnostics, especially in oncological applications.     

Bimodal paramagnetic and fluorescent liposomes for cellular and tumor magnetic resonance imaging

A novel bimodal fluorescent and paramagnetic liposome is described for cellular labeling. In this study, we show the synthesis of a novel gadolinium lipid, Gd.DOTA.DSA, designed for liposomal cell labeling and tumor imaging. Liposome formulations consisting of this lipid were optimized in order to allow for maximum cellular entry, and the optimized formulation was used to label HeLa cells in vitro. The efficiency of this novel bimodal Gd-liposome formulation for cell labeling was demonstrated using both fluorescence microscopy and magnetic resonance imaging (MRI). The uptake of Gd-liposomes into cells induced a marked reduction in their MRI T 1 relaxation times. Fluorescence microscopy provided concomitant proof of uptake and revealed liposome internalization into the cell cytosol. The optimized formulation was also found to exhibit minimal cytotoxicity and was shown to have capacity for plasmid DNA (pDNA) transfection. A further second novel neutral bimodal Gd-liposome is described for the labeling of xenograft tumors in vivo utilizing the enhanced permeation and retention effect (EPR). Balb/c nude mice were inoculated with IGROV-1 cells, and the resulting tumor was imaged by MRI using these in vivo Gd-liposomes formulated with low charge and a poly(ethylene glycol) (PEG) calyx for long systemic circulation. These Gd-liposomes which were less than 100 nm in size were shown to accumulate in tumor tissue by MRI, and this was also verified by fluorescence microscopy of histology samples. Our in vivo tumor imaging results demonstrate the effectiveness of MRI to observe passive targeting of long-term circulating liposomes to tumors in real time, and allow for MRI directed therapy, wherein the delivery of therapeutic genes and drugs to tumor sites can be monitored while therapeutic effects on tumor mass and/or size may be simultaneously observed, quantitated, and correlated.     

单克隆抗体ND-1-量子点荧光探针对大肠癌细胞的特异成像研究

目的制备抗人大肠癌单克隆抗体ND-1的量子点荧光探针,实现对大肠癌细胞的靶向成像。方法采用共价偶联方法,以1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(NHS)为缩合剂,通过在反应体系中加入不同摩尔比例的单克隆抗体ND-1和游离量子点QD605进行条件优化,制备偶联产物ND-1-QD605荧光探针;利用荧光光谱扫描技术对ND-1-QD605进行光学特性表征,并检测其抗光漂白能力;利用免疫荧光方法检测ND-1-QD605对大肠癌细胞的靶向结合能力。结果在量子点QD605与单克隆抗体ND-1摩尔比1:40条件下,可实现二者的高效偶联;荧光光谱分析显示ND-1-QD605保留了游离量子点QD605优良的荧光特性;在激发光照射1h内,ND-1-QD605荧光强度未发生明显改变;荧光显微镜观察可见该探针能够与表达有相应抗原LEA的人大肠癌CCL187细胞特异性结合,呈现高灵敏度、特异性荧光成像。结论制备的单克隆抗体ND-1的量子点荧光探针具有大肠癌细胞靶向成像能力,有望为大肠癌的体内靶向成像研究和临床诊断提供新方法。     

Application of quantum dots as probes for correlative fluorescence, conventional, and energy-filtered transmission electron microscopy.

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide-ranging applications for cell imaging. The electron density and elemental composition of these materials permit the extension of their use as probes in conventional electron microscopy (TEM) and energy-filtered TEM (EFTEM). Here we illustrate the feasibility of using streptavidin-conjugated QDs as TEM tags by labeling a nuclear protein on cell sections and obtaining correlative fluorescence and TEM data. We also show that QD probes can be employed in conjunction with immunogold for co-localization of proteins at the ultrastructural level. Furthermore, by obtaining cadmium elemental maps of CdSe/ZnS QDs distributed on a nuclear structure, we demonstrate the potential of QDs for co-localization of multiple proteins when used in combination with EFTEM.     

Dynamic imaging of lymphatic vessels and lymph nodes using a bimodal nanoparticulate contrast agent

BACKGROUND: Evaluation of lymphedema and lymph node metastasis in humans has relied primarily on invasive or radioactive modalities. While noninvasive technologies such as magnetic resonance imaging (MRI) offer the potential for true three-dimensional imaging of lymphatic structures, invasive modalities, such as optical fluorescence microscopy, provide higher resolution and clearer delineation of both lymph nodes and lymphatic vessels. Thus, contrast agents that image lymphatic vessels and lymph nodes by both fluorescence and MRI may further enhance our understanding of the structure and function of the lymphatic system. Recent applications of bimodal (fluorescence and MR) contrast agents in mice have not achieved clear visualization of lymphatic vessels and nodes. Here the authors describe the development of a nanoparticulate contrast agent that is taken up by lymphatic vessels to draining lymph nodes and detected by both modalities. METHODS: A unique nanoparticulate contrast agent composed of a polyamidoamine dendrimer core conjugated to paramagnetic contrast agents and fluorescent probes was synthesized. Anesthetized mice were injected with the nanoparticulates in the hind footpads and imaged by MR and fluorescence microscopy. High resolution MR and fluorescence images were obtained and compared to traditional techniques for lymphatic visualization using Evans blue dye. RESULTS: Lymph nodes and lymphatic vessels were clearly observed by both MRI and fluorescence microscopy using the bimodal nanoparticulate contrast agent. Characteristic tail-lymphatics were also visualized by both modalities. Contrast imaging yielded a higher resolution than the traditional method employing Evans blue dye. MR data correlated with fluorescence and Evans blue dye imaging. CONCLUSION: A bimodal nanoparticulate contrast agent facilitates the visualization of lymphatic vessels and lymph nodes by both fluorescence microscopy and MRI with strong correlation between the two modalities. This agent may translate to applications such as the assessment of malignancy and lymphedema in humans and the evaluation of lymphatic vessel function and morphology in animal models.