For the last several years, members of the Center for Molecular Microscopy have been developing techniques for 3D high resolution electron microscopic imaging of cells and tissues. These techniques include focused ion beam scanning electron microscopy (FIB-SEM), as well as other complementary methods. As this technology has matured, the laboratory has developed interest in several key biological research areas, while still maintaining active technology development research.
Cellular Imaging Technology
A number of tools are emerging that promise to provide nanoscale information about cells and tissues; these tools include a number of super resolution light microscopy techniques, as well as 3D electron microscopy techniques. At the Center for Molecular Microscopy, our primary techniques include focused ion beam scanning electron microscopy (FIB-SEM, otherwise known as ion abrasion scanning electron microscopy, or IA-SEM), correlated with standard confocal or super resolution light microscopy.
FIB-SEM imaging requires that a sample be fixed, either chemically or by high-pressure freezing, stained with heavy metals, and embedded in resin. The FIB-SEM microscope images by scanning the face of a resin-embedded block with the electron beam, then removing a thin (> 5 nm) layer of material with an ion beam, and then imaging again. By alternating imaging and material removal, this technique produces a stack of 2D images, which can then be reconstructed into 3D. Segmentation and analysis of this data can reveal the 3D shapes of many ultrastructural features of a cell or tissue, including cell membranes, cellular organelles, or even viral particles.
Because traditional FIB-SEM imaging does not allow for imaging of specific probes, we are currently investigating a number of techniques to identify the localization of proteins or structures of interest. Some of these techniques include correlative fluorescent and electron microscopic imaging, electron-dense protein tags, as well as techniques that combine traditional immunolabeling for electron microscopy with 3D imaging.
Areas of Biological Research Interest
Our initial work with FIB-SEM focused on two basic areas: understanding the architecture of cell-cell interactions, primarily in the context of virological synapses, and the ultrastructure of subcellular organelles in a variety of cell and tissue types.
Our recent work on HIV-1 virological synapses has included the study of HIV-1 in macrophages, immature and mature dendritic cells, T cells, and astrocytes. The use of FIB-SEM, which allows the direct visualization of cellular structural features and individual virions in all three dimensions, was key to discovering a number of key features of HIV-1 cell-to-cell transfer. One major feature of HIV-1 virological synapses is the highly complex three-dimensional interactions between the membranes of neighboring cells. For example, within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in dendritic cells. In addition, FIB-SEM directly revealed pockets of HIV virions within macrophages that are contiguous with the extracellular space; similarly, this technique showed complex membrane interactions between infected and uninfected T cells, including both tight cellular clustering as well as long filopodial extensions.
A new area of interest for the Center for Molecular Microscopy include the structural aspects of metabolism and energy; this is reflected both in the protein structure part of our program, as well as in the cellular imaging area. Recent work in this area has included 3D structural analysis of mitochondria in skeletal muscle cells. Another new area of interest for us is the structural mechanisms of cell motility, cell migration, and the relevance of these to metastasis.