Improved diffuse fluorescence flow cytometer prototype for high sensitivity detection of rare circulating cells in vivo

Abstract. Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×103 cells/mL. In combination, these improvements resulted in an overall cell counting sensitivity of about 1 cell/mL or better in vivo

Singlet oxygen luminescence as an in vivo photodynamic therapy dose metric: validation in normal mouse skin with topical amino-levulinic acid

Although singlet oxygen (1O2) has long been proposed as the primary reactive oxygen species in photodynamic therapy (PDT), it has only recently been possible to detect it in biological systems by its luminescence at 1270 nm. Having previously demonstrated this in vitro and in vivo, we showed that cell survival was strongly correlated to the 1O2 luminescence in cell suspensions over a wide range of treatment parameters. Here, we extend this to test the hypothesis that the photobiological response in vivo is also correlated with 1O2 generation, independent of individual treatment parameters. The normal skin of SKH1-HR hairless mice was sensitised with 20% amino-levulinic acid-induced protoporophyrin IX and exposed to 5, 11, 22 or 50 J cm−2 of pulsed 523 nm light at 50 mW cm−2, or to 50 J cm−2 at 15 or 150 mW cm−2. 1O2 luminescence was measured during treatment and the photodynamic response of the skin was scored daily for 2 weeks after treatment. As observed by other authors, a strong irradiance dependence of the PDT effect was observed. However, in all cases the responses increased with the 1O2 luminescence, independent of the irradiance, demonstrating for the first time in vivo an unequivocal mechanistic link between 1O2 generation and photobiological response

Mechanisms for Tuning Engineered Nanomaterials to Enhance Radiation Therapy of Cancer.

Engineered nanomaterials that produce reactive oxygen species on exposure to X- and gamma-rays used in radiation therapy offer promise of novel cancer treatment strategies. Similar to photodynamic therapy but suitable for large and deep tumors, this new approach where nanomaterials acting as sensitizing agents are combined with clinical radiation can be effective at well-tolerated low radiation doses. Suitably engineered nanomaterials can enhance cancer radiotherapy by increasing the tumor selectivity and decreasing side effects. Additionally, the nanomaterial platform offers therapeutically valuable functionalities, including molecular targeting, drug/gene delivery, and adaptive responses to trigger drug release. The potential of such nanomaterials to be combined with radiotherapy is widely recognized. In order for further breakthroughs to be made, and to facilitate clinical translation, the applicable principles and fundamentals should be articulated. This review focuses on mechanisms underpinning rational nanomaterial design to enhance radiation therapy, the understanding of which will enable novel ways to optimize its therapeutic efficacy. A roadmap for designing nanomaterials with optimized anticancer performance is also shown and the potential clinical significance and future translation are discussed

Photosensitizer Drug Delivery via an Optical Fiber

: An optical fiber has been developed with a maneuverable miniprobe tip that sparges O2 gas and photodetaches pheophorbide (sensitizer) molecules. Singlet oxygen is produced at the probe tip surface which reacts with an alkene spacer group releasing sensitizer upon fragmentation of a dioxetane intermediate. Optimal sensitizer photorelease occurred when the probe tip was loaded with 60 nmol sensitizer, where crowding of the pheophorbide molecules and self-quenching were kept to a minimum. The fiber optic tip delivered pheophorbide molecules and singlet oxygen to discrete locations. The 60 nmol sensitizer was delivered into petrolatum; however, sensitizer release was less efficient in toluene-d8 (3.6 nmol) where most had remained adsorbed on the probe tip, even after the covalent alkene spacer bond had been broken. The results open the door to a new area of fiber optic-guided sensitizer delivery for the potential photodynamic therapy of hypoxic structures requiring cytotoxic control

Computed Tomography Imaging of Primary Lung Cancer in Mice Using a Liposomal-Iodinated Contrast Agent

To investigate the utility of a liposomal-iodinated nanoparticle contrast agent and computed tomography (CT) imaging for characterization of primary nodules in genetically engineered mouse models of non-small cell lung cancer.Primary lung cancers with mutations in K-ras alone (Kras(LA1)) or in combination with p53 (LSL-Kras(G12D);p53(FL/FL)) were generated. A liposomal-iodine contrast agent containing 120 mg Iodine/mL was administered systemically at a dose of 16 µl/gm body weight. Longitudinal micro-CT imaging with cardio-respiratory gating was performed pre-contrast and at 0 hr, day 3, and day 7 post-contrast administration. CT-derived nodule sizes were used to assess tumor growth. Signal attenuation was measured in individual nodules to study dynamic enhancement of lung nodules.A good correlation was seen between volume and diameter-based assessment of nodules (R(2)>0.8) for both lung cancer models. The LSL-Kras(G12D);p53(FL/FL) model showed rapid growth as demonstrated by systemically higher volume changes compared to the lung nodules in Kras(LA1) mice (p<0.05). Early phase imaging using the nanoparticle contrast agent enabled visualization of nodule blood supply. Delayed-phase imaging demonstrated significant differential signal enhancement in the lung nodules of LSL-Kras(G12D);p53(FL/FL) mice compared to nodules in Kras(LA1) mice (p<0.05) indicating higher uptake and accumulation of the nanoparticle contrast agent in rapidly growing nodules.The nanoparticle iodinated contrast agent enabled visualization of blood supply to the nodules during the early-phase imaging. Delayed-phase imaging enabled characterization of slow growing and rapidly growing nodules based on signal enhancement. The use of this agent could facilitate early detection and diagnosis of pulmonary lesions as well as have implications on treatment response and monitoring

Quantum dot loaded immunomicelles for tumor imaging

<p>Abstract</p> <p>Background</p> <p>Optical imaging is a promising method for the detection of tumors in animals, with speed and minimal invasiveness. We have previously developed a lipid coated quantum dot system that doubles the fluorescence of PEG-grafted quantum dots at half the dose. Here, we describe a tumor-targeted near infrared imaging agent composed of cancer-specific monoclonal anti-nucleosome antibody 2C5, coupled to quantum dot (QD)-containing polymeric micelles, prepared from a polyethylene glycol/phosphatidylethanolamine (PEG-PE) conjugate. Its production is simple and involves no special equipment. Its imaging potential is great since the fluorescence intensity in the tumor is twofold that of non-targeted QD-loaded PEG-PE micelles at one hour after injection.</p> <p>Methods</p> <p>Para-nitrophenol-containing (5%) PEG-PE quantum dot micelles were produced by the thin layer method. Following hydration, 2C5 antibody was attached to the PEG-PE micelles and the QD-micelles were purified using dialysis. 4T1 breast tumors were inoculated subcutaneously in the flank of the animals. A lung pseudometastatic B16F10 melanoma model was developed using tail vein injection. The contrast agents were injected via the tail vein and mice were depilated, anesthetized and imaged on a Kodak Image Station. Images were taken at one, two, and four hours and analyzed using a methodology that produces normalized signal-to-noise data. This allowed for the comparison between different subjects and time points. For the pseudometastatic model, lungs were removed and imaged <it>ex vivo </it>at one and twenty four hours.</p> <p>Results</p> <p>The contrast agent signal intensity at the tumor was double that of the passively targeted QD-micelles with equally fast and sharply contrasted images. With the side views of the animals only tumor is visible, while in the dorsal view internal organs including liver and kidney are visible. <it>Ex vivo </it>results demonstrated that the agent detects melanoma nodes in a lung pseudometastatic model after a 24 hours wash-out period, while at one hour, only a uniform signal is detected.</p> <p>Conclusions</p> <p>The targeted agent produces ultrabright tumor images and double the fluorescence intensity, as rapidly and at the same low dose as the passively targeted agents. It represents a development that may potentially serve to enhance early detection for metastases.</p

Comparison of fluorescence tomographic imaging in mice with early-arriving and quasi-continuous-wave photons.

The highly diffuse nature of light propagation in biological tissue is a major challenge for obtaining high-fidelity fluorescence tomographic images. In this work we investigated the use of time-gated detection of early-arriving photons for reducing the effects of light scatter in mice relative to quasi-cw photons. When analyzing sinographic representations of the measured data, it was determined that early photons allowed a reduction in the measured FWHM of fluorescent targets by a factor of approximately 2-3, yielding a significant improvement in the tomographic image reconstruction quality

Elucidating structure and function in vivo with hybrid fluorescence and magnetic resonance imaging.

While the mathematics, physics, and technology behind magnetic resonance (MR) and fluorescence image formation are distinctively different, the two modalities have significant complementary features to impart strong preclinical and clinical application synergies. Traditionally, hybrid MR and fluorescence imaging implied the use of a system where optical and MR signals can be concurrently acquired. In this case, the common geometry allows for the superposition of fluorescence images of cellular and subcellular processes onto anatomical and functional MR images. More recently, a different hybrid imaging paradigm is strongly evolving by utilizing hybrid MR-fluorescence nanoparticles. This approach offers a second paradigm of hybrid visualization where the common underlying contrast enables the coregistration of MR and fluorescence images acquired under different geometries. We review herein progress with the evolving field of multimodality MR and fluorescence imaging and discuss how these strategies offer a highly promising outlook in established and in novel preclinical and clinical applications

Complete angle small animal fluorescence imaging with early-arriving photons.

Fluorescence mediated tomography allows quantitative, three-dimensional imaging of optical reporter probes in whole animals and is therefore emerging as a powerful molecular imaging tool. The achievable image quality in fluorescence tomography is limited by the high-degree of light scatter in biological tissue. Time-gated detection of early-arriving and therefore minimally-scattered photons transmitted through diffusive tissue is one strategy for minimizing the effects of light scatter. In this work, we performed full-angle tomographic imaging of mice implanted with fluorescent tubes using time-gated detection of early- and later-arriving photons. This was achieved using a femtosecond laser and a high-speed, time-gated intensified CCD imager. We demonstrate that the early-transmitted fluorescent photons allow improved visualization of the fluorescence distribution, even when considering individual projections through the animal. High-fidelity image reconstruction using 72 projections in 5-degree steps using early-arriving photons is also demonstrated