Technology Development

A key part of our research program is the development of novel technologies for high resolution, 3D imaging of biological materials. We are actively engaged in technology development in both the Cellular Imaging and Cryo-EM sections of our research program.

 

New Technologies for Cellular Imaging

Our primary technology for cellular imaging is focused ion beam scanning electron microscopy (FIB-SEM), a technique that allows serial imaging of fixed, resin-embedded biological materials. Our recent development work with this technology has included several key features that allow for more stable, targeted imaging of areas of interest, as well as techniques for correlating light microscopy with the 3D data acquired with FIB-SEM.

The first of these features is a method for tracking imaging in the z-direction; by depositing a precisely marked protective layer of carbon and platinum, one can both reduce charging and imaging artifacts while simultaneously automatically determining the accurate depth between individual 2D images in the stack.

The second feature, “keyframe imaging”, allows for selective high resolution imaging within a larger, low-resolution volume. Because FIB-SEM images can be very large, and imaging a large area at high resolution is time-intensive, keyframe imaging is designed to shorten the data acquisition process. This feature allows a researcher to automatically select regions of interest, and follow and adjust high-resolution imaging areas during data acquisition.

Although FIB-SEM imaging allows the visualization of heavy-metal stained ultrastructural features of cells and tissues, the technique does not currently allow visualization of specific labeled proteins. Thus correlation of FIB-SEM volumes with light microscopy of labeled proteins allows one to localize these proteins to specific features of interest.

Current areas of interest for technology development for cellular imaging include new, enhanced algorithms for correlation between FIB-SEM and light or super resolution microscopy, as well as novel methods for FIB-SEM compatible tags and labels. Additionally, we are also exploring new methods for sample preparation, and finding new applications for FIB-SEM in imaging and analysis of biological materials.

 

New Technologies for Cryo-EM

Our work in the cryo-EM field is highly focused on finding new ways to improve resolution in protein structure determination, and in discovering ways to directly image proteins and stuctures within the cellular context.

Recent developments from our lab have been centered primarily in the use of sub-tomogram averaging to determine protein structures from within larger tomographic datasets. This has been particularly useful for the determination of the structures of envelope glycoprotein spikes directly from the surface of HIV, influenza and Ebola viral particles.

We have also actively pursued methods to better streamline and improve image collection by cryo-electron microscopy. In this technique, acquiring a large number of high quality images is a time- and resource-consuming process; we are interested in finding ways to quantitatively evaluate images for quality in real time, allowing a microscope to “intelligently” select imaging areas and discard poor images.

Finally, structural heterogeneity is a common problem in protein structure determination; the presence of heterogeneity can lower resolution significantly. We are also looking at ways to computationally separate a spectrum of structural conformations, thus both increasing the quality of structures and revealing key mechanistic detail about protein function.