Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes ~90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take ~30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine

Structural analysis of multicellular organisms with cryo-electron tomography

We developed a method for visualizing tissues from multicellular organisms using cryo-electron tomography. Our protocol involves vitrifying samples with high-pressure freezing, thinning them with cryo-FIB-SEM (focused-ion-beam scanning electron microscopy) and applying fiducial gold markers under cryogenic conditions to the lamellae post-milling. We applied this protocol to acquire tomograms of vitrified Caenorhabditis elegans embryos and worms, which showed the intracellular organization of selected tissues at particular developmental stages in otherwise intact specimens