Nicole Y. Morgan

In vivo multiple color lymphatic imaging using upconverting nanocrystals

Upconverting nanocrystals are unique nano-sized particles that emit light at shorter wavelengths (visible and near infrared) after excitation in the near infrared that dramatically reduces background autofluorescence in in vivo two color lymphatic imaging for depicting the lymphatic channels and nodes.

Interpretation of dark-field contrast and particle-size selectivity in grating interferometers

In grating-based x-ray phase sensitive imaging, dark-field contrast refers to the extinction of the interference fringes due to small-angle scattering. For configurations where the sample is placed before the beamsplitter grating, the dark-field contrast has been quantified with theoretical wave propagation models. Yet when the grating is placed before the sample, the dark-field contrast has only been modeled in the geometric optics regime. Here we attempt to quantify the dark-field effect in the grating-before-sample geometry with first-principle wave calculations and understand the associated particle-size selectivity. We obtain an expression for the dark-field effect in terms of the sample materials complex refractive index, which can be verified experimentally without fitting parameters. A dark-field computed tomography experiment shows that the particle-size selectivity can be used to differentiate materials of identical x-ray absorption.

Immune complexes stimulate CCR7-dependent dendritic cell migration to lymph nodes

Antibodies are critical for defense against a variety of microbes, but they may also be pathogenic in some autoimmune diseases. Many effector functions of antibodies are mediated by Fcγ receptors (FcγRs), which are found on most immune cells, including dendritic cells (DCs)—important antigen-presenting cells that play a central role in inducing antigen-specific tolerance or immunity. Following antigen acquisition in peripheral tissues, DCs migrate to draining lymph nodes via the lymphatics to present antigen to T cells. Here we demonstrate that FcγR engagement by IgG immune complexes (ICs) stimulates DC migration from peripheral tissues to the paracortex of draining lymph nodes. In vitro, IC-stimulated mouse and human DCs showed greater directional migration in a chemokine (C-C) ligand 19 (CCL19) gradient and increased chemokine (C-C) receptor 7 (CCR7) expression. Using intravital two-photon microscopy, we observed that local administration of IC resulted in dermal DC mobilization. We confirmed that dermal DC migration to lymph nodes depended on CCR7 and increased in the absence of the inhibitory receptor FcγRIIB. These observations have relevance to autoimmunity because autoantibody-containing serum from humans with systemic lupus erythematosus (SLE) and from a mouse model of SLE also increased dermal DC migration in vivo, suggesting that this process may occur in lupus, potentially driving the inappropriate localization of autoantigen-bearing DCs.

Nanoscintillator Conjugates as Photodynamic Therapy-Based Radiosensitizers: Calculation of Required Physical Parameters

Abstract Morgan, N. Y., Kramer-Marek, G., Smith, P. D., Camphausen, K. and Capala, J. Nanoscintillator Conjugates as Photodynamic Therapy-Based Radiosensitizers: Calculation of Required Physical Parameters. Radiat. Res. 171, 236–244 (2009). The recent demonstration of nanoscale scintillators has led to interest in the combination of radiation and photodynamic therapy. In this model, scintillating nanoparticles conjugated to photosensitizers and molecular targeting agents would enhance the targeting and improve the efficacy of radiotherapy and extend the application of photodynamic therapy to deeply seated tumors. In this study, we calculated the physical parameters required for these nanoparticle conjugates to deliver cytotoxic levels of singlet oxygen at therapeutic radiation doses, drawing on the published literature from several disparate fields. Although uncertainties remain, it appears that the light yield of the nanoscintillators, the efficiency of energy transfer to the photosensitizers, and the cellular uptake of the nanoparticles all need to be fairly well optimized to observe a cytotoxic effect. Even so, the efficacy of the combination therapy will likely be restricted to X-ray energies below 300 keV, which limits the application to brachytherapy.

Motionless phase stepping in X-ray phase contrast imaging with a compact source

Significance From diagnostic exams to security screening, a major concern in X-ray imaging is the potential damage from absorbed radiation energy. Phase contrast techniques are being developed to alleviate the concern by detecting the slight refractive bending of X-rays in an object, instead of relying on the attenuation of the beam. A front runner in the development is technologies that require mechanical scanning of a grating in the X-ray beam to attain high-resolution images. This paper reports a motionless, electromagnetic scanning method in place of mechanical scanning. It lifts the constraints on speed and flexibility and reduces the complexity and cost of the technologies, all of which help bring them closer to everyday applications. X-ray phase contrast imaging offers a way to visualize the internal structures of an object without the need to deposit significant radiation, and thereby alleviate the main concern in X-ray diagnostic imaging procedures today. Grating-based differential phase contrast imaging techniques are compatible with compact X-ray sources, which is a key requirement for the majority of clinical X-ray modalities. However, these methods are substantially limited by the need for mechanical phase stepping. We describe an electromagnetic phase-stepping method that eliminates mechanical motion, thus removing the constraints in speed, accuracy, and flexibility. The method is broadly applicable to both projection and tomography imaging modes. The transition from mechanical to electromagnetic scanning should greatly facilitate the translation of X-ray phase contrast techniques into mainstream applications.

Subnanoradian X-ray phase-contrast imaging using a far-field interferometer of nanometric phase gratings

Hard X-ray phase-contrast imaging characterizes the electron density distribution in an object without the need for radiation absorption. The power of phase contrast to resolve subtle changes, such as those in soft tissue structures, lies in its ability to detect minute refractive bending of X-rays. Here we report a far-field, two-arm interferometer based on the new nanometric phase gratings, which can detect X-ray refraction with subnanoradian sensitivity, and at the same time overcomes the fundamental limitation of ultra-narrow bandwidths (Δλ/λ~10−4) of the current, most sensitive methods based on crystal interferometers. On a 1.5% bandwidth synchrotron source, we demonstrate clear visualization of blood vessels in unstained mouse organs in simple projection views, with over an order-of-magnitude higher phase contrast than current near-field grating interferometers.

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture

Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100μm drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions.

Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors

Summary Pluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

A Portable Fluorescence Camera for Testing Surgical Specimens in the Operating Room: Description and Early Evaluation

PurposeClinical translation of novel optical probes requires testing of human specimens ex vivo to ensure efficacy. However, it may be difficult to remove human tissue from the operating room due to regulatory/privacy issues. Therefore, we designed a portable fluorescence camera to test targeted optical imaging probes on human specimens in the operating room.ProceduresA compact benchtop fluorescence camera was designed and built in-house. A mouse xenograft model of ovarian cancer with an activatable imaging probe based on rhodamine green was used to test the device. Comparison was made to commercially available imaging systems.ResultsThe prototype camera produced images comparable to images acquired with commercially available, non-portable imaging systems.ConclusionWe demonstrate the feasibility of a specimen-based portable fluorescence camera for use in the operating room. Its small size ensures that tissue excised from patients can be tested promptly for fluorescence within the operating room environment, thus expediting the testing of novel imaging probes.

Grazing angle Mach-Zehnder interferometer using reflective phase gratings and a polychromatic, un-collimated light source

Normal incidence Talbot-Lau interferometers in x-ray applications have the drawbacks of low fringe visibility with polychromatic sources when the wave propagation distance is increased to achieve higher phase sensitivity, and when fabrication limits the attainable grating density. In contrast, reflective gratings illuminated at grazing angles have dramatically higher effective densities than their physical values. However, new designs are needed for far field interferometers using grazing angle geometry with incoherent light sources. We show that, with the appropriate design and choice of reflective phase gratings, there exist pairs of interfering pathways of exactly equal lengths independent of the incoming beams incidence angle and wavelength. With a visible light grazing angle Mach-Zehnder interferometer, we show the conditions for achieving near ideal fringe visibility and demonstrate both absolute and differential phase-contrast imaging. We also describe the design parameters of an x-ray interferometer and key factors for its implementation.