Development of an x-ray method for cell tracking using gold nanoparticles

2009/2010Cell tracking is the capability to locate the position and to follow the fate of cells in living beings over a long period of time. This method might have a major impact on different sectors of medicine and biology, such as regenerative medicine or tumor research. Cell tracking permits a better understanding of cellular processes such as cell migration, cell homing, and cell function in engraftments at organ or tissue level, or the dynamics of tumor growth and metastatic spread. Due to the combination of classical imaging techniques and cellular contrast agents mainly based on nanotechnologies cell tracking is already feasible. Successful cell tracking has been carried out using magnetic resonance imaging in combination with superparamagnetic iron oxide nanoparticles, optical imaging combined with quantum dots or positron emission computed tomography using unstable β+ nuclides. Although outstanding results have been achieved in specific cases utilizing these techniques, none of them can be really considered an all-embracing cell tracking technique since each of them features specific drawbacks. Despite of the ubiquitous application of x-ray imaging methods, up to now, x-rays have been rarely considered as a cell tracking solution because of radiation dose constrains. The thesis at hand tries to exploit x-ray imaging methods in the field of cell tracking. Starting from the x-ray properties an appropriate cellular contrast agent, in this case colloidal gold nano particles, and subsequent a loading protocol based on phagocytosis has been chosen and tested. Expected signal levels, contrasts and signal to noise ratios have been theoretically determined for this specific marker and loading protocol. Then, the detectability of marked cells has been verified on in vitro and ex vivo models proofing the feasibility of cell tracking using gold nanoparticles in combination with x-ray CT. Successively radiation dose constrains have been examined in detail by means of computer simulations and low dose ex vivo experiments. The investigation revealed that in vivo cell tracking using x-rays is feasible and on the basis of these results in vivo experiments had been designed and prepared. All the necessary authorizations had been obtained by the National and Local Ethic Committees. First successful in vivo cell tracking experiments in animal models of human diseases have been performed during the last months of this thesis work. The results presented in the following reveal that also x-ray cell tracking encompasses some limitations such as the previous mentioned techniques, however it can be consider an alternative and attractive method for cell tracking.XXIII Ciclo198

Increased material differentiation through multi-contrast x-ray imaging: a preliminary evaluation of potential applications to the detection of threat materials

Most material discrimination in security inspections is based on dual-energy x-ray imaging, which enables the determination of a material's effective atomic number (Z eff) as well as electron density and its consequent classification as organic or inorganic. Recently phase-based "dark-field" x-ray imaging approaches have emerged that are sensitive to complementary features of a material, namely its unresolved microstructure. It can therefore be speculated that their inclusion in the security-based imaging could enhance material discrimination, for example of materials with similar electron densities and Z eff but different microstructures. In this paper, we present a preliminary evaluation of the advantages that such a combination could bear. Utilising an energy-resolved detector for a phase-based dark-field technique provides dual-energy attenuation and dark-field images simultaneously. In addition, since we use a method based on attenuating x-ray masks to generate the dark-field images, a fifth (attenuation) image at a much higher photon energy is obtained by exploiting the x-rays transmitted through the highly absorbing mask septa. In a first test, a threat material is imaged against a non-threat one, and we show how their discrimination based on maximising their relative contrast through linear combinations of two and five imaging channels leads to an improvement in the latter case. We then present a second example to show how the method can be extended to discrimination against more than one non-threat material, obtaining similar results. Albeit admittedly preliminary, these results indicate that significant margins of improvement in material discrimination are available by including additional x-ray contrasts in the scanning process

Asymmetric masks for laboratory-based X-ray phase-contrast imaging with edge illumination

We report on an asymmetric mask concept that enables X-ray phase-contrast imaging without requiring any movement in the system during data acquisition. The method is compatible with laboratory equipment, namely a commercial detector and a rotating anode tube. The only motion required is that of the object under investigation which is scanned through the imaging system. Two proof-of-principle optical elements were designed, fabricated and experimentally tested. Quantitative measurements on samples of known shape and composition were compared to theory with good agreement. The method is capable of measuring the attenuation, refraction and (ultra-small-angle) X-ray scattering, does not have coherence requirements and naturally adapts to all those situations in which the X-ray image is obtained by scanning a sample through the imaging system

Utility of knife-edge position tracking in cycloidal computed tomography

Cycloidal computed tomography provides high-resolution images within relatively short scan times by combining beam modulation with dedicated under-sampling. However, implementing the technique relies on accurate knowledge of the sample’s motion, particularly in the case of continuous scans, which is often unavailable due to hardware or software limitations. We have developed an easy-to-implement position tracking technique using a sharp edge, which can provide reliable information about the trajectory of the sample and thus improve the reconstruction process. Furthermore, this approach also enables the development of other innovative sampling schemes, which may otherwise be difficult to implement

Early post-metamorphic, Carboniferous blastoid reveals the evolution and development of the digestive system in echinoderms

Inferring the development of the earliest echinoderms is critical to uncovering the evolutionary assembly of the phylum-level body plan but has long proven problematic because early ontogenetic stages are rarely preserved as fossils. Here, we use synchrotron tomography to describe a new early post-metamorphic blastoid echinoderm from the Carboniferous (approx. 323 Ma) of China. The resulting three-dimensional reconstruction reveals a U-shaped tubular structure in the fossil interior, which is interpreted as the digestive tract. Comparisons with the developing gut of modern crinoids demonstrate that crinoids are an imperfect analogue for many extinct groups. Furthermore, consideration of our findings in a phylogenetic context allows us to reconstruct the evolution and development of the digestive system in echinoderms more broadly; there was a transition from a straight to a simple curved gut early in the phylum's evolution, but additional loops and coils of the digestive tract (as seen in crinoids) were not acquired until much later

X-ray phase-contrast microtomography of soft tissues using a compact laboratory system with two-directional sensitivity

X-ray microtomography is a nondestructive, three-dimensional inspection technique applied across a vast range of fields and disciplines, ranging from research to industrial, encompassing engineering, biology, and medical research. Phase-contrast imaging extends the domain of application of x-ray microtomography to classes of samples that exhibit weak attenuation, thus appearing with poor contrast in standard x-ray imaging. Notable examples are low-atomic-number materials, like carbon-fiber composites, soft matter, and biological soft tissues. We report on a compact and cost-effective system for x-ray phase-contrast microtomography. The system features high sensitivity to phase gradients and high resolution, requires a low-power sealed x-ray tube, a single optical element, and fits in a small footprint. It is compatible with standard x-ray detector technologies: in our experiments, we have observed that single-photon counting offered higher angular sensitivity, whereas flat panels provided a larger field of view. The system is benchmarked against known-material phantoms, and its potential for soft-tissue three-dimensional imaging is demonstrated on small-animal organs: a piglet esophagus and a rat heart. We believe that the simplicity of the setup we are proposing, combined with its robustness and sensitivity, will facilitate accessing quantitative x-ray phase-contrast microtomography as a research tool across disciplines, including tissue engineering, materials science, and nondestructive testing in general

Fusarium oxysporum f.sp. radicis-lycopersici induces distinct transcriptome reprogramming in resistant and susceptible isogenic tomato lines

8openInternationalItalian coauthor/editorBackground: Fusarium oxysporum f.sp. radicis-lycopersici (FORL) is one of the most destructive necrotrophic pathogens affecting tomato crops, causing considerable field and greenhouse yield losses. Despite such major economic impact, little is known about the molecular mechanisms regulating Fusarium oxysporum f.sp. radicis-lycopersici resistance in tomato. Results: A transcriptomic experiment was carried out in order to investigate the main mechanisms of FORL response in resistant and susceptible isogenic tomato lines. Microarray analysis at 15 DPI (days post inoculum) revealed a distinct gene expression pattern between the two genotypes in the inoculated vs non-inoculated conditions. A model of plant response both for compatible and incompatible reactions was proposed. In particular, in the incompatible interaction an activation of defense genes related to secondary metabolite production and tryptophan metabolism was observed. Moreover, maintenance of the cell osmotic potential after the FORL challenging was mediated by a dehydrationinduced protein. As for the compatible interaction, activation of an oxidative burst mediated by peroxidases and a cytochrome monooxygenase induced cell degeneration and necrosis. Conclusions: Our work allowed comprehensive understanding of the molecular basis of the tomato-FORL interaction. The result obtained emphasizes a different transcriptional reaction between the resistant and the susceptible genotype to the FORL challenge. Our findings could lead to the improvement in disease control strategies.openManzo, D.; Ferriello, F.; Puopolo, G.; Zoina, A.; D’Esposito, D.; Tardella, L.; Ferrarini, A.; Ercolano, M.R.Manzo, D.; Ferriello, F.; Puopolo, G.; Zoina, A.; D’Esposito, D.; Tardella, L.; Ferrarini, A.; Ercolano, M.R

Micropropagación de caña de azúcar en Ecuador

Ecuador is a country where sugar cane (Saccharum spp. hybrid) exceeds 82 000 ha for sugar production. The Research Center of the National Union of sugar cane farming in Ecuador (UNCE) is plotted as objective to obtain plants of different varieties by micropropagation, to multiply quickly and efficiently, genotypes with excellent performance under the conditions of production and result in a high quality certified seed and plant genetics. It were used as initial explants shoots, of the varieties ‘Cenicaña 1985-1992’ (CC85-92), ‘Ragnar’, ‘BJ 7046’, ‘CR 74-250’, ‘SP70-1143’, ‘PCG 12 745’ ‘RD7511’ and ‘Mexico 73-0523’. The obtained seedlings were multiplied, rooted and acclimatised. Then be planted in areas where sugar cane is grown in the country. In the case of the variety ‘Ragnar’ planted an area of approximately 0.3 ha, which was determined yield (ton/ha). Micropropagation of selected varieties allowed to obtain 268 600 plants in 12 months. It was found that the varieties showed differences in their rates of multiplication. The survival of plants in the acclimatization phase was higher than 88.0%. The harvest for the experimental plot planted with the variety ‘Ragnar’ contributed 55 tons. This equates to an output of 183 ton/ha. These values are much higher than the national average. The results confirmed the non-appearance of diseased plants or Scalding RSD. These results are one of the first reports for Ecuador from the use of biotechnological methods, being the pioneers in the mass propagation of sugarcane plants.Keywords: mass propagation, Saccharum spp. hybrid, seedEcuador es un país donde la caña de azúcar (Saccharum spp. híbrido) supera las 82 000 ha dedicadas a la producción de azúcar. El Centro de Investigación de la Unión Nacional de Cañicultores del Ecuador (UNCE) se trazó como objetivo obtener plantas de diferentes variedades por micropropagación, que permitiera multiplicar de manera rápida y eficaz, genotipos con excelente comportamiento en las condiciones de producción y que resultaran en una semilla certificada de alta calidad genética y fitosanitaria. Se emplearon como explantes iniciales meristemos apicales de las variedades ‘CeniCaña 85-92’ (CC85-92),‘Ragnar’, ‘BJ 7046’, ‘CR 74-250’, ‘SP70-1143’, ‘PCG 12745’, ‘RD7511’ y ‘México 73-0523’. Las plántulas obtenidas se multiplicaron, enraizaron y aclimatizaron. Posteriormente, se sembraron en áreas donde se cultiva caña de azúcar en el país. En el caso de la variedad ‘Ragnar’ se plantó un área de aproximadamente 0.3 ha, en la cual se determinó el rendimiento (t/ha). La micropropagación de las variedades seleccionadas permitió obtener 268 600 plantas en 12 meses. Se comprobó que las variedades mostraron diferencias en sus coeficientes de multiplicación. La supervivencia de las plantas en la fase de aclimatización fue superior al 88.0%. La cosecha correspondiente a la parcela experimental sembrada con la variedad ‘Ragnar’ aportó 55 t. esto equivale a una producción de 183 t/ha. Estos valores son muy superiores a la media nacional. Los resultados confirmaron la no aparición de plantas enfermas por RSD o Escaldadura. Estos resultados constituyen uno de los primeros informes para Ecuador de la utilización de métodos biotecnológicos, siendo los pioneros en la propagación masiva de plantas de caña de azúcar.Palabras clave: propagación masiva, Saccharum spp. híbrido, semill