New Findings about Cryo-Electron Tomography (Cryo-ET) You May Want to Know

Posted by Jerry Carter on August 4th, 2021

On July 8, 2021, Journal of Structural Biology published online the technological innovation achievement "VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography" in collaboration with Sun Fei's research group by the Biological Imaging Center of Protein Science Research Platform, Institute of Biophysics, Chinese Academy of Sciences, and developed VHUT-cryo-FIB, a technological innovation after the cell sample frozen water-containing section preparation technology developed by the center in 2016, expanding the research object of in situ structural biology from single cells to tissue samples closer to physiological states.

Cryo-electron tomography (cryo-ET) is an important cryo-EM technique that can obtain in situ three-dimensional high-resolution ultrastructure of cell and tissue samples, in situ structural information of biomacromolecules, and in situ interaction information of protein machines. The technology is considered to be a bridge between molecular biology and cell biology and is known as "visual proteomics". However, this technique requires that the thickness of the sample must be below 300 nm, and thinner samples (below 150 nm) are required to obtain high-resolution information, but most biological samples are above several microns in thickness and cannot be directly studied using this technique. In addition, in order to study the structure of biological tissue samples closer to the physiological state, it is usually necessary to use high-pressure freezing technology to freeze and fix biological tissue samples, but the thickness of samples after high-pressure freezing is generally more than 100 μm. How to prepare high-quality biological tissue sample sections suitable for cryoelectron tomography research is an important technical problem facing the field of in situ structural biology.

Based on the latest cryo-focused ion beam technology (cryo-FIB), a set of cryo-transmission hardware is designed and developed in this technical study, which effectively combines vibrating slice technology, high-pressure freezing technology, cryo-trimming technology and cryo-focused ion beam thinning technology, innovates the cryo-focused ion beam cutting process, and forms a complete and efficient technical process for the preparation of frozen water-containing slices of tissue samples VHUT-cryo-FIB. Frozen aqueous sections of tissue samples between 150 and 300 nm in thickness can be efficiently prepared using this technical process. In this study, frozen aqueous sections of spinach leaves, mouse skeletal muscle, liver, and myocardium were prepared using the VHUT-cryo-FIB method, respectively, and the in situ three-dimensional structures of spinach cytoplasmic ribosomes (34 Å) and mouse liver cytoplasmic ribosomes (18 Å) were successfully solved. These results demonstrate that the VHUT-cryo-FIB method can be widely used in frozen aqueous section preparation of various biological tissue samples, providing a powerful sample preparation method for in situ structural biology studies.

Collected by Creative Biostructure, that is equipped with state-of-the-art electron microscopes and expertise in cryo-ET. We are always determined to working with our clients closely to solve problems, which will benefit the complete understanding of cell biology and drug design and development in the long run. Moreover, Creative Biostructure has extensive experience in using cryo-EM technology to study dynamic conformational changes and function of macromolecular complexes and has successfully applied this technology to characterize the structures of gene therapy and drug delivery particles.