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 recently developed tools, including a variety of super resolution light microscopy techniques and 3D electron microscopy techniques, are capable of providing nanoscale information about cells and tissues. 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), often in conjunction with correlative light microscopy, to image large samples in 3D and at nanoscale resolutions.
In FIB-SEM imaging, a sample is fixed chemically or by high-pressure freezing, stained with heavy metals, and then embedded in resin. In the FIB-SEM instrument, a thin (~ 5 nm) layer of material is ablated or milled away with an ion beam, revealing a polished face of the resin-embedded sample; this face is imaged with the scanning electron beam, and a subsequent layer milled again. By alternating imaging and milling, this technique produces a stack of 2D EM images, which can then be computationally reconstructed into a 3D image volume. 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.
Currently, we are 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. We are also working on hardware and software improvements that will allow for artifact-free imaging of larger biological samples.