The integrity of the basement membrane is essential for tissue cellular growth and is often altered in disease. In this work a method for noninvasively detecting the structural integrity of the basement membrane, based on the delivery of cationic iron-oxide nanoparticles, was developed. Cationic particles accumulate due to the highly negative charge of proteoglycans in the basement membrane. The kidney was used to test this technique because of its highly fenestrated endothelia and wellestablished disease models to manipulate the basement membrane charge barrier.
The subventricular zone (SVZ) is a continual source of neural progenitors throughout adulthood. Many of the animal models designed to study the migration of these cells from the ventricle to places of interest like the olfactory bulb or an injury site require histology to localize precursor cells. Here, it is demonstrated that up to 30% of the neural progenitors that migrate along the rostral migratory stream (RMS) in an adult rodent can be labeled for MRI via intraventricular injection of micron sized particles of iron oxide (MPIOs). The precursors migrating from the SVZ along the RMS were found to populate the olfactory bulb with all three types of neural cells; neurons, oligodendrocytes, and astrocytes. In all cases 10-30% of these cells were labeled in the RMS en route to the olfactory bulb. Ara-C an anti-mitotic agent eliminated precursor cells at the SVZ, RMS, and olfactory bulb and also eliminated the MRI detection of the precursors. This indicates that MRI signal detected is due to progenitor cells that leave the SVZ and is not due to non-specific diffusion of MPIOs. Using MRI to visualize neural progenitor cell behavior in individual animals during plasticity or disease models should be a useful tool, especially in combination with other information that MRI can supply.
The development and characterisation of a fluorescent optical PEBBLE (Probe Encapsulated By Biologically Localised Embedding) nanosensor for the detection of zinc is detailed. A ratiometric sensor has been fabricated that incorporates two fluorescent dyes; one is sensitive to zinc and the other acts as a reference. The sensing components are entrapped within a polymer matrix by a microemulsion polymerisation process that produces spherical sensors that are in the size region of 20 to 200 nm. Cellular measurements are made possible by the small sensor size and the biocompatibility of the matrix. The effects of reversibility, photobleaching and leaching have been examined, as well as the selectivity towards zinc over other cellular ions such as Na+, Ca2+, K+, and Mg2+. The dynamic range of these sensors was found to be 4 to 50 microM Zn2+ with a linear range from 15 to 40 microM. The response time for the PEBBLE is less than 4 s and the sensor is reversible. In addition, the nanosensors are photostable and leaching from the matrix, determined using a novel method, is minimal. These sensors are capable of real-time inter- and intra-cellular imaging and are insensitive to interference from proteins.
Molecular Probes' Alexa Fluor dyes are generally used for biological labeling because of their ideal fluorescent properties, but here we detail Alexa Fluor 488's nanomolar sensitivity to free iron. Furthermore, the dye has been encapsulated into a polymer nanosphere by a microemulsion method, producing <100 nm particles. These nanosensors, PEBBLEs (Probe Encapsulated By Biologically Localized Embedding) have micromolar sensitivity and are non-responsive to other metal ions of biological interest.
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