Alignment of muscle precursor cells on the vertical edges of thick carbon nanotube films.

The development of scaffolds and templates is an essential aspect of tissue engineering. We show that thick (>0.5 mm) vertically aligned carbon nanotube films, made by chemical vapour deposition, can be used as biocompatible substrates for the directional alignment of mouse muscle cells where the cells grow on the exposed sides of the films. Ultra high resolution scanning electron microscopy reveals that the films themselves consist mostly of small diameter (10 nm) multi-wall carbon nanotubes of wavy morphology with some single wall carbon nanotubes. Our findings show that for this alignment to occur the nanotubes must be in pristine condition. Mechanical wiping of the films to create directional alignment is detrimental to directional bioactivity. Larger areas for study have been formed from a composite of multiply stacked narrow strips of nanotubes wipe-transferred onto elastomer supports. These composite substrates appear to show a useful degree of alignment of the cells.

Site-specific 3D imaging of cells and tissues with a dual beam microscope.

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.