Self‐illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano‐particle

Autofluorescence arising from normal tissues can compromise the sensitivity and specificity of in vivo fluorescence imaging by lowering the target‐to‐background signal ratio. Since bioluminescence resonance energy transfer quantum dot (BRET‐QDot) nano‐particles can self‐illuminate in near‐infrared in the presence of the substrate, coelenterazine, without irradiating excitation lights, imaging using BRET‐QDots does not produce any autofluorescence. In this study, we applied this BRET‐QDot nano‐particle to the in vivo lymphatic imaging in mice in order to compare with BRET, fluorescence or bioluminescence lymphatic imaging. BRET‐QDot655, in which QDot655 is contained as a core, was injected at different sites (e.g. chin, ear, forepaws and hind paws) in mice followed by the intravenous coelenterazine injection, and then bioluminescence and fluorescence imaging were serially performed. In all mice, each lymphatic basin was clearly visualized in the BRET imaging with minimal background signals. The BRET signal in the lymph nodes lasted at least 30 min after coelenterazine injections. Furthermore, the BRET signal demonstrated better quantification than the fluorescence signal emitting from QDot655, the core of this BRET particle. These advantages of BRET‐QDot allowed us to perform real‐time, quantitative lymphatic imaging without image processing. BRET‐Qdots have the potential to be a robust nano‐material platform for developing optical molecular imaging probes. Copyright © 2010 John Wiley & Sons, Ltd.     

Use of colloidal quantum dots as a digitally switched swept light source for gold nanoparticle based hyperspectral microscopy

We propose a method to utilize colloidal quantum dots (QDs) as a swept light source for hyperspectral microscopy. The use of QD allows for uniform multicolor emission which covers visible-NIR wavelengths. We used 8 colors of CdSe/ZnS and CdTe/ZnS colloidal quantum dots with the peak emission wavelengths from 520 nm to 800 nm. The QDs are packed in a compact enclosure, composing a low-cost, solid-state swept light source that can be easily used in most microscopes. Multicolor emission from the QDs is simply controlled by digitally switching excitation UVLEDs, eliminating the use of mechanically-driven gratings or filters. We used gold nanoparticles as optical markers for hyperspectral microscopy. Due to the effect of localized surface plasmon resonance, gold nanoparticles demonstrate size and shape-dependent absorption spectra. Employed in a standard microscope, the QD light source enabled multispectral absorption imaging of macrophage cells labeled with gold nanorods and nanospheres.OCIS codes: (170.0110) Imaging systems, (110.4234) Multispectral and hyperspectral imaging, (300.6550) Spectroscopy, visible, (170.1530) Cell analysis, (160.4236) Nanomaterials, (230.3670) Light-emitting diodes     

Spectral near‐infrared fluorescence imaging of curved surfaces using projection reconstruction algorithms

In vivo spectral fluorescence imaging has made it possible to non‐invasively visualize superficial curved structures as well as structures deep to the skin. However, the defocus created by blurring has been an obstacle to creating anatomically interpretable surface images. Herein we present a methodology to correct for blurring induced by curved structures during spectral fluorescence imaging using signal intensity projection algorithms. In a phantom and an animal model in which the lymphatic system was visualized after the interstitial injection of quantum dots with emission spectra in the near‐infrared (NIR) range, the planes of focus were sequentially adjusted to obtain a z‐stack of images which contains images acquired from multiple focal points. Maximum, minimum, median and average intensity projections were applied to the resulting images. Using the phantom, the minimum and the median intensity projection images demonstrated improved deblurring whereas during in vivo imaging the median intensity projection images more clearly visualized important structures than did the other projection techniques. Image stacking with subsequent application of appropriate projection techniques provides a simple method for deblurring in vivo optical images obtained from curved surfaces, thus improving their anatomic resolution. Copyright © 2007 John Wiley & Sons, Ltd.     

Fluorescence lifetime imaging of activatable target specific molecular probes

In vivo optical imaging using fluorescently labeled self‐quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target‐to‐background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self‐quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2⁺ and BALB/3T3 (HER2^?) cell lines. Changes in fluorescence lifetime correlated with temperature‐ and time‐dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2⁺ and BALB/3T3 (HER2^?)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target‐specific activatable antibody–fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

基于量子点标记技术在人血清甲胎蛋白中的意义

目的建立1种基于新型纳米颗粒——量子点(QD)作为荧光标志物检测人血清甲胎蛋白的新方法。方法生物素标记的甲胎蛋白单克隆抗体与血清中的甲胎蛋白抗原特异性结合形成抗原抗体复合物,耦联生物素的QD-605和QD-655作为荧光标志物与抗原抗体复合物结合,借助磁场的清洗分离效应,去除反应体系中的非特异性物质,利用荧光分光光度计检测QD标记的甲胎蛋白抗原抗体复合物的荧光强度及光谱。结果用350nm波长的光作为激发光,量子点QD-605单独作为标志物时,荧光发射谱在605nm处有1个波峰;QD-655单独作为标志物时,荧光发射谱在655nm处有1个波峰;QD-605和QD-655混合作为标志物时,荧光发射谱出现2个波峰,分别在605和655nm处。结论本选题利用QD标记技术检测血清中的甲胎蛋白,有望为临床免疫诊断学提供1种更快捷、有效的方法。     

Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level.     

Multimodal optical studies of single and clustered colloidal quantum dots for the long-term optical property evaluation of quantum dot-based molecular imaging phantoms

Understanding the optical properties of clustered quantum dots (QDs) is essential to the design of QD-based optical phantoms for molecular imaging. Single and clustered core/shell colloidal QDs of dimers, trimers, and tetramers are self-assembled, separated, and preferentially collected using electrospray differential mobility analysis (ES-DMA) with electrostatic deposition. Multimodal optical characterization and analysis of their dynamical photoluminescence (PL) properties enables the long-term evaluation of the physicochemical and optical properties of QDs in a single or a clustered state. A multimodal time-correlated spectroscopic confocal microscope capable of simultaneously measuring the time evolution of PL intensity fluctuation, PL lifetime, and emission spectra reveals the long-term dynamic optical properties of interacting QDs in individual dimeric clusters of QDs. This new method will benefit research into the quantitative interpretation of fluorescence intensity and lifetime results in QD-based molecular imaging techniques. The process of photooxidation leads to coupling of the QDs in a dimer, leading to unique optical properties when compared to an isolated QD. These results guide the design and evaluation of QD-based phantom materials for the validation of the PL measurements for quantitative molecular imaging of biological samples labeled with QD probes.     

Pixel-based absorption correction for dual-tracer fluorescence imaging of receptor binding potential

Ratiometric approaches to quantifying molecular concentrations have been used for decades in microscopy, but have rarely been exploited in vivo until recently. One dual-tracer approach can utilize an untargeted reference tracer to account for non-specific uptake of a receptor-targeted tracer, and ultimately estimate receptor binding potential quantitatively. However, interpretation of the relative dynamic distribution kinetics is confounded by differences in local tissue absorption at the wavelengths used for each tracer. This study simulated the influence of absorption on fluorescence emission intensity and depth sensitivity at typical near-infrared fluorophore wavelength bands near 700 and 800 nm in mouse skin in order to correct for these tissue optical differences in signal detection. Changes in blood volume [1-3%] and hemoglobin oxygen saturation [0-100%] were demonstrated to introduce substantial distortions to receptor binding estimates (error > 30%), whereas sampled depth was relatively insensitive to wavelength (error < 6%). In response, a pixel-by-pixel normalization of tracer inputs immediately post-injection was found to account for spatial heterogeneities in local absorption properties. Application of the pixel-based normalization method to an in vivo imaging study demonstrated significant improvement, as compared with a reference tissue normalization approach.OCIS codes: (170.3660) Light propagation in tissues, (170.3880) Medical and biological imaging, (170.4580) Optical diagnostics for medicine     

BRET based dual-colour (visible/near-infrared) molecular imaging using a quantum dot/EGFP–luciferase conjugate

Owing to its high sensitivity, bioluminescence imaging is an important tool for biosensing and bioimaging in life sciences. Compared to fluorescence imaging, bioluminescence imaging has a superior advantage that the background signals resulting from autofluorescence are almost zero. In addition, bioluminescence imaging can permit long-term observation of living cells because external excitation is not needed, leading to no photobleaching and photocytotoxicity. Although bioluminescence imaging has such superior properties over fluorescence imaging, observation wavelengths in bioluminescence imaging are mostly limited to the visible region. Here we present bioluminescence resonance energy transfer (BRET) based dual-colour (visible/near-infrared) molecular imaging using a quantum dot (QD) and luciferase protein conjugate. This bioluminescent probe is designed to emit green and near-infrared luminescence from enhanced green fluorescent protein (EGFP) and CdSeTe/CdS (core/shell) QDs, where EGFP–Renilla luciferase (RLuc) fused proteins are conjugated to the QDs. Since the EGFP–RLuc fused protein contains an immunoglobulin binding domain (GB1) of protein G, it is possible to prepare a variety of molecular imaging probes functionalized with antibodies (IgG). We show that the BRET-based QD probe can be used for highly sensitive dual-colour (visible/near-infrared) bioluminescence molecular imaging of membrane receptors in cancer cells.

A bioluminescent dual-colour molecular-imaging probe was prepared to emit green and near-infrared luminescence from a conjugate between enhanced green fluorescent protein (EGFP), Renilla luciferase (RLuc) and CdSeTe/CdS quantum dot (QD).     

In vivo local fluorescence spectroscopy in PDD of superficial bladder cancer

ObjectiveA combination of fluorescence imaging with in vivo local fluorescence spectroscopy (LFS) was used to improve the predictive ability of photodynamic diagnosis (PDD) of superficial bladder cancer after intravesical instillation of Alasense, a 5-aminolevulinic acid (5-ALA)-based agent.Material and methodA total of 62 patients with superficial malignancies of the urinary bladder were included in a preliminary clinical study. Nineteen patients underwent autofluorescence (AF) examination, whereas 43 patients underwent photodynamic diagnosis (PDD) after intravesical instillation of Alasense (NIOPIK, Russia). After visual examination of the urinary bladder wall under white and blue light, fluorescence emission spectra were recorded in vivo under 442 and 532 nm laser excitation within visible red fluorescent zones that had been preliminarily revealed by fluorescence imaging. For spectral data interpretation, a spectral fluorescence parameter was introduced as a ratio of the 5-ALA-induced protoporphyrin IX-emission intensity to that of the AF emission. In the search for a quantitative criterion for this parameter, as well as a diagnostic algorithm to facilitate differentiation between inflammatory and neoplastic tissues, two approaches were tested based on: (1) a posteriori estimation of the threshold values of the spectral fluorescence parameters for inflammation and cancerous tissues, and (2) a posteriori probability of attributing an acquired emission spectrum to a histologically confirmed tissue type.ResultsIn the group of patients without intravesical instillation of Alasense (n=19), LFS showed a dramatic drop (with a factor of 5–20, p<0.0001) in the AF emission intensity of superficial bladder malignancies under 442 and 532 nm laser excitation, although no reliable distinction was revealed in the AF emission intensity between normal urothelium and inflammatory foci. In the group of patients with intravesical instillation of Alasense (n=43), false positive (FP) results were confirmed histologically in 18 of 54 fluorescent foci revealed by fluorescence imaging, and the positive predictive value (PPV) was estimated to be 67%. Correlations of the spectral fluorescence parameter with the histological results were studied and probability distribution functions (PDFs) of this parameter were constructed for normal urothelium (type I), inflammation, papilloma, low-grade dysplasia (type II), as well as high-grade dysplasia, CIS and TCC (type III) at both excitation wavelengths. PDF distributions for “inflammation – neoplasia” were shown to have essential overlaps, causing certain problems in definition of the thresholds of the spectral fluorescence parameters and attribution of the recorded spectra to the three tissue types. Despite this, the threshold-based approach allowed for a reduction in the number of FP cases to 3 and an increase of the PPV to 91%. In another approach, the maximum realization probabilities of the spectral fluorescence parameter were selected as criteria for attribution of the tested tissue to one of the histological tissue types. Estimations based on this approach showed that the probability of FP cases and the PPV could be roughly estimated as 0–0.1 and as 91–100% at λ_ex=442 and 532 nm respectively.ConclusionThis preliminary clinical study shows that both approaches allow for a significant increase in the PPV of PDD of superficial bladder cancer. Therefore the combination of fluorescence imaging with in vivo LFS may be helpful for minimizing false-positive fluorescence and reducing the number of biopsies necessary.     

Feasibility,sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.     

Silver ion-doped CdTe quantum dots as fluorescent probe for Hg2+ detection

Mercury(ii), which is a well-known toxic species, exists in the industrial waste water in many cases. In the present work, CdTe quantum dots (QDs) are studied as a fluorescence probe for Hg²⁺ detection. Ag ions are induced to QDs to enlarge their detection concentration range. l-cysteine is employed in the QD-based fluorescence probe to connect QDs with Hg²⁺. X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy results indicate the formation of zinc blende CdTe QDs with sizes of ∼5 nm and the existence of Ag⁺ in crystalline CdTe. Photoluminescence (PL) spectra and PL decay spectra were acquired to investigate the emission mechanism of Ag-doped CdTe QDs, revealing multi-emission in QD samples with higher concentrations of Ag⁺ doping. The highest PL quantum yield of the QD samples was 59.4%. Furthermore, the relationship between the fluorescence intensity and the concentration of Hg²⁺ has been established. Two linear relationships were obtained for the plot of F/F₀ against Hg²⁺ concentration, enlarging the detection concentration range of Hg²⁺.

Ag-doped CdTe QDs emit multiple-fluorescence peaks, and the relationship between fluorescence intensity and the concentration of Hg²⁺ is established. Two linear relationships are obtained, which is benefit to the extension of detection range.     

两种量子点标记用于抗核抗体检测的对比研究与评价

目的对比研究两种不同粒径量子点(QDs)标记抗体在抗核抗体(ANA)检测中的成像效果,评价其在临床样本检测中的应用价值。方法 Hep-2细胞为抗原片,分别以QD655和QD605标记第二抗体(二抗),用间接免疫荧光分析(IIFA)检测114例自身免疫病患者和30例健康对照者血清ANA,以异硫氰酸荧光素(FITC)标记的二抗为对照。单特异性ANA用免疫印迹法(IBT)确认。比较和评价各法应用效果。结果经对114例患者血清研究发现,两种QD标记二抗均可见与FITC标记二抗检测ANA结果相同或类似的荧光模式,但ANA滴度略显不同,对核颗粒型与胞浆颗粒型ANA测定灵敏度以QD655为最高,QD605次之,FITC标记二抗相对较弱。而对核均质型、核仁型与核着丝点型ANA测定灵敏度高低依次为FITC、QD655和QD605标记二抗。相关分析结果显示,两种QD与FITC标记物检测ANA滴度结果均呈高度正相关,r值分别为0.834、0.832(P均<0.01)。两种QD标记二抗对核颗粒型、胞浆颗粒型荧光模式与FITC标记二抗检测结果阳性符合率均为100%;而核均质型与核仁型检测,阳性结果符合率为80.6%～84.4%。30例用FITC标记二抗检测ANA阴性血清中,两种QD标记二抗检测均见1例ANA阳性。两种QD与FITC标记物检测ANA总符合率均超过90.0%,有高度的一致性(Kappa值均>0.75)。结论 QD655和QD605标记二抗用于ANA检测总体结果稳定、灵敏、可行;QDs粒径越大其标记二抗测定ANA灵敏度越高,但对某些低滴度免疫荧光核型(包括核均质型、核仁型与核着丝点型)ANA有漏检可能。     

Molecular imaging of tumors with nanobodies and antibodies: Timing and dosage are crucial factors for improved in vivo detection

The utility of nanobodies and conventional antibodies for in vivo imaging is well known, but optimum dosing and timing schedules for one versus the other have not been established. We aimed to improve specific tumor imaging in vivo with nanobodies and conventional antibodies using near‐infrared fluorescence (NIRF) imaging. We used ARTC2 expressed on lymphoma cells as a model target antigen. ARTC2‐specific nanobody s+16a and conventional antibody Nika102 were labeled with NIRF‐dye AF680. In vivo NIRF‐imaging of ARTC2‐positive and ARTC2‐negative xenografts was performed over 24 h post‐injection of 5, 10, 25, or 50 µg of each conjugate. Specific target‐binding and tissue‐penetration were verified by NIRF imaging ex vivo, flow cytometry and fluorescence microscopy. NIRF‐imaging of s+16a⁶⁸⁰ in vivo revealed a six times faster tumor accumulation than of Nika102⁶⁸⁰. Using 50 µg of s+16a⁶⁸⁰ increased the specific signals of ARTC2‐positive tumors without increasing background signals, allowing a tumor‐to‐background (T/B) ratio of 12.4 ± 4.2 within 6 h post‐injection. Fifty micrograms of Nika102⁶⁸⁰ increased specific signals of ARTC2‐positive tumors but also of ARTC2‐negative tumors and background, thereby limiting the T/B ratio to 6.1 ± 2.0. Ten micrograms of Nika102⁶⁸⁰ only slightly reduced specific tumor signals but dramatically reduced background signals. Ex vivo analyses confirmed a faster and deeper tumor penetration with s+16a⁶⁸⁰. Using nanobody s+16a allowed same‐day imaging with a high T/B ratio, whereas antibody Nika102 gave optimal imaging results only 24 h post injection. Nanobody s+16a required a high dose, whereas antibody Nika102 had the best T/B‐ratio at a low dose. Therefore, timing and dosage should be addressed when comparing nanobodies and conventional antibodies for molecular imaging purposes. Copyright © 2015 John Wiley & Sons, Ltd.     

A fluorescent chromophore TOTO‐3 as a ‘smart probe’ for the assessment of ultrasound‐mediated local drug delivery in vivo

Many potent anti‐cancer drugs have an intracellular mode of action, but are limited in crossing the cell membrane, resulting in a reduced clinical efficacy. Ultrasound (US) is known to facilitate the penetration of drugs into tumors cells. However (molecular) imaging techniques that monitor in vivo the underlying processes of US‐triggered drug delivery are lacking. The objective of this study was to demonstrate the feasibility of using a fluorescent nuclear acid stain (TOTO‐3) as a model drug to monitor in real‐time US‐mediated delivery by in vivo fluorescence imaging. Following co‐injection of TOTO‐3 and microbubbles US was applied to the tumor. The time course of the drug delivery process was monitored by fluorescence imaging. Immunohistological analysis and in vitro experiments were performed to investigate the results in more detail. A significant signal intensity enhancement of the US‐treated tumor was observed that indicates intracellular delivery of the dye. In the control tumor TOTO‐3 signal was strongly associated with macrophages, which was not the case for the sonicated tumor. The capability of macrophages to uptake TOTO‐3 was confirmed in vitro. This study demonstrates that an optical contrast agent with similar characteristics to an anti‐cancer drug may be used for continuous in vivo monitoring of the drug delivery process. Copyright © 2010 John Wiley & Sons, Ltd.     

A radiolabeled nonapeptide probe targeting PC‐3 cells and bone metastases of prostate cancer in mice

Previous investigations showed that interleukin‐11 (IL‐11) and the IL‐11 receptor (IL‐11R) are correlated with regulation of tumor progression and may play significant roles in bone metastases. The nonapeptide structure c(CGRRAGGSC) is a phage‐display‐selected IL‐11 mimic that binds to IL‐11R. The aim of this study was to synthesize radiolabeled c(CGRRAGGSC) and to investigate the possible interaction between this radioactive probe and an IL‐11R‐positive bone metastasis model of PC‐3 prostate cancer. The molecular probe ^99mTc–DTPA–c(CGRRAGGSC) was radiolabeled with ^99mTc using the diethylenetriaminepentaacetic acid (DTPA) chelate. Counterstaining was performed with LSS670, a near‐infrared dye. The binding sites of the molecular probe in PC‐3 cells were observed under a fluorescence microscope. The binding characteristics of the labeled probe were analyzed using radioreceptor analysis. Single photon emission tomography imaging and biodistribution of the probe were investigated using xenografts of PC‐3 cells into tibias of nude mice. The labeled product, ^99mTc–DTPA–c(CGRRAGGSC), was obtained with high labeling efficiency, high radiochemical purity and good stability. The molecular probe was combined with the PC‐3 cell membrane and cytoplasm through fluorescence tracing. In the saturation and competitive inhibition experiments performed in vitro, the K_d value was 0.32 ± 0.02 n m and the B_max value was 754 ± 34 fmol mg^?1 pro. The probe exhibited a high tumor uptake in vivo. The radioactive molecular probe ^99mTc–DTPA–c(CGRRAGGSC) may be used as a specific molecular imaging agent for detecting IL‐11R overexpression in tumors and bone metastasis, such as prostate cancers. Copyright © 2012 John Wiley & Sons, Ltd.     

Non‐invasive in vivo tracking of fibrin degradation by fluorescence imaging

Fibrin‐based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid‐phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non‐invasive molecular imaging systems enable real‐time tracking of processes in the living animal. In this study, a non‐invasive fluorescence based imaging system was applied to follow and quantify site‐specific degradation of fibrin sealant. To enable non‐invasive tracking of fibrin in vivo, fibrin‐matrix was labelled by incorporation of a fluorophore‐conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore‐labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo real‐time lymphatic draining using quantum‐dot optical imaging in mice

The lymphatic system is essential for fluid regulation and for the maintenance of host immunity. However, in vivo lymph flow is difficult to track in real time, because of the lack of an appropriate imaging method. In this study, we combined macro‐zoom fluorescence microscopy with quantum‐dot (Qdot) optical lymphatic imaging to develop an in vivo real‐time optical lymphatic imaging method that allows the tracking of lymph through lymphatic channels and into lymph nodes. After interstitial injection of Qdots in a mouse, rapid visualization of the cervical lymphatics and cervical lymph nodes was achieved. Real‐time monitoring of the injected Qdots revealed that the cortex of the node enhanced first followed by a net‐like pattern in the central portion of the node. Histology revealed that the rim and net‐like enhancing regions corresponded to the subcapsular sinuses and medullary sinuses respectively. Additionally, multiplexed two‐color real‐time lymphatic tracking was performed with two different Qdots. With this real‐time imaging system, we successfully tracked microscopic lymphatic flow in vivo. This method could have a potential impact for lymphatic research in visualizing normal or abnormal functional lymphatic flows. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Nature‐inspired nanoformulations for contrast‐enhanced in vivo MR imaging of macrophages

Magnetic resonance imaging (MRI) of macrophages in atherosclerosis requires the use of contrast‐enhancing agents. Reconstituted lipoprotein particles that mimic native high‐density lipoproteins (HDL) are a versatile delivery platform for Gd‐based contrast agents (GBCA) but require targeting moieties to direct the particles to macrophages. In this study, a naturally occurring methionine oxidation in the major HDL protein, apolipoprotein (apo) A‐I, was exploited as a novel way to target HDL to macrophages. We also tested if fully functional GBCA–HDL can be generated using synthetic apo A‐I peptides. The fluorescence and MRI studies reveal that specific oxidation of apo A‐I or its peptides increases the in vitro macrophage uptake of GBCA–HDL by 2–3 times. The in vivo imaging studies using an apo E‐deficient mouse model of atherosclerosis and a 3.0 T MRI system demonstrate that this modification significantly improves atherosclerotic plaque detection using GBCA–HDL. At 24 h post‐injection of 0.05 mmol Gd kg^?1 GBCA–HDL containing oxidized apo A‐I or its peptides, the atherosclerotic wall/muscle normalized enhancement ratios were 90 and 120%, respectively, while those of GBCA–HDL containing their unmodified counterparts were 35 and 45%, respectively. Confocal fluorescence microscopy confirms the accumulation of GBCA–HDL containing oxidized apo A‐I or its peptides in intraplaque macrophages. Together, the results of this study confirm the hypothesis that specific oxidation of apo A‐I targets GBCA–HDL to macrophages in vitro and in vivo. Furthermore, our observation that synthetic peptides can functionally replace the native apo A‐I protein in HDL further encourages the development of these contrast agents for macrophage imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

MR and optical imaging of early micrometastases in lymph nodes: triple labeling with nano‐sized agents yielding distinct signals

Few imaging methods are available for depicting in vivo cancer cell migration within the lymphatic system. Detection of such early micrometastases requires extremely high target to background. In this study, we dual‐labeled human breast cancer cells (MDA‐MB468) with a small particle of iron oxide (SPIO) and a quantum dot (QD), and tracked these cells in the lymphatic system in mice using in vivo MRI and optical imaging. A generation‐6 gadolinium‐dendrimer‐based MRI contrast agent (Gd‐G6) was employed for visualizing regional lymphatic channels and nodes. Since Gd‐G6 shortened T₁ leading to high signal, whereas SPIO‐labeled cancer cells greatly lowered signal, a small number of cells were simultaneously visualized within the draining lymphatic basins. One million dual‐labeled cancer cells were subcutaneously injected into the paws of mice 24 h prior to imaging. Then whole body images were acquired pre‐ and post‐intracutaneous injection of Gd‐G6 with 3D‐T₁w‐FFE and balanced‐FFE sequences for cancer cell tracking and MR lymphangiography. In vivo MRI clearly visualized labeled cancer cells migrating from the paw to the axillary lymph nodes using draining lymphatics. In vivo optical imaging using a fluorescence surgical microscope demonstrated tiny cancer cell clusters in the axillary lymph node with high spatial resolution. Thus, using a combination of MRI and optical imaging, it is possible to depict macro‐ and early micrometastases within the lymphatic system. This platform offers a versatile research tool for investigating and treating lymphatic metastases in animal models. Copyright © 2011 John Wiley & Sons, Ltd.