Dual-beam slice/view tomography for cellular/tissue 3D architecture

The dual-beam slice/view tomography technique involves serial sectioning of biological specimen with the ion beam and imaging of each section with the electron beam (secondary electron imaging) in the dual-beam microscopes. This tomography technique bridges the gaps between confocal microscopy and TEM tomography. It offers advantages of large volume (up to hundreds of micrometers) and site-specific 3D viewing of cellular and tissue structure with the resolution in nanometer size range. Therefore, it is a powerful method for studying 3D architecture of cellular and tissue structures.

fig1. 3D visualization of Trypanosome Brucei parasite

For biological samples, the major challenges for slice/view tomography lie in large penetration depth of e-beam in low-Z biological samples and the radiation damage for both e-beam and ion-beam. The former affects the resolution of secondary electron imaging due to signal delocalization, while the later results in the sample damage and structural disordering which will also finally affect the imaging resolution. To achieve high-resolution 3D reconstruction, both sample preparation and imaging/milling parameters play key roles. Good sample preparation not only keeps the original cellular/tissue structures but also ensures effective staining of the nanometer sized structures as interested.  Tailoring the milling parameters can minimize the structure disordering induced by ion bombardment/implantation, and employing suitable imaging conditions can reduce the radiation damage (radiolysis damage) and signal delocalization to guarantee imaging resolution.

This project is intended to

1. develop good sample preparation protocols for dual-beam slice/view technique and
2. explore the capabilities of our advanced dual-beam electron microscopy for high-resolution 3D reconstruction of cellular/tissue architectures with the collaboration with PIs in CBIS.
   

   fig2.3D visualization of microvilli in mice intestine

Currently we are working with Ass/Prof. Cynthia He for trypanosome brucei parasite samples and exploring the possibility of using the 3D reconstruction method to study the complex microtubule structures in this parasite.

Team

• Paul Matsudaira, PI
• Victor Horodincu, Co-PI
• Cynthia He, Co-PI
• Binghai Liu, for dual-beam slice and view, and 3D imaging process
• Ying Sun, for EM sample preparation

Tech

• FEI Helios Nanolab 600 Dual-Beam System for slice/view

fig. 1 3D visualization of Trypanosome Brucei parasite by dual-beam slice/view technique (the 3D view was reconstructed by Amira)

fig. 2 3D visualization of microvilli in mice intestine by dual-beam slice/view technique (the 3D view was reconstructed by Amira)