Sustainable Developments in Eliminating Charging and Correlative Multi-modal Imaging techniques for Serial Block-face Scanning Electron Microscopy
- Author(s): Johnson, Donald Dewane
- Advisor(s): Ellisman, Mark H
- Taylor, Susan S
- et al.
Serial block-face scanning electron microscopy (SBEM) promises to revolutionize structural biology and neuroanatomical research by allowing the 3-dimensional reconstruction of relatively large regions of tissue and cell arrays at near nanometer-scale resolution. This approach employs an automated ultra-microtome fitted into a scanning electron microscope that images the specimen surface, or blockface, following each iterative nanometer thin cut. However, a principal limitation of this approach is the resolution obtainable using backscatter electrons at low accelerating voltages due to the build up of electrostatic charges on the block-face, otherwise known as "charging." Herein, we present a specimen preparation protocol that implements heavy metal staining, and a series of methods for use of conductive materials, as either a dopant, covalent linker, or metal coordinated matrix (scaffold), in the epoxy resin. These approaches in staining and enhancing resin conductivity lead to substantial improvement in contrast and image resolution for accelerating voltages at, or below, 2.0keV for SBEM. To build connections from the 3-dimensional data sets obtained by SBEM to other imaging modalities for broader context and insight, developments in region of interest (ROI) tracking across light, xray and electron microscopy using upconverting nanoparticles, as fiducial markers and labels, have led to advanced efficiency in the correlated microscopy workflow. As a result, a universal fiducial/marker ties together the representing datasets for multi-modal imaging, and adds further context to the specimen's region of interest.