Cryo-correlative light and electron microscopy (cryo-CLEM) transfer holder for high-resolution imaging

Project Summary Project Title: Cryo-correlative light and electron microscopy (cryo-CLEM) transfer holder for high- resolution imaging Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Khim Karki, Ph.D. Summary: Cryogenic transmission electron microscopy (cryo-TEM) technique has enabled important structural and dynamic information of complex cells or macro-molecules by freezing the samples in their native environment. However, biological materials suffer from poor contrast in TEM and requires pre-screening of the regions of interest (ROI) using fluorescence marker and/or beads. The sample grid is transferred into the TEM without disturbing the pre-mapped coordinates for correlative imaging – the workflow commonly referred to as cryogenic cross-correlative light and electron microscopy (cryo-CLEM) technique. Currently, there is no commercial solution that allows interrupted transfers of sample between different cryo-CLEM tools such as focused ion beam (FIB), light microscopy (LM) and TEM, all while maintaining the cryogenic temperatures (andlt; -140°C) of the sample. High-yield and high-throughput analysis is also key to identifying structures in several dimensions and orientations (e.g. proteins), especially when using conventional TEM approaches. The use of multiple sample transfer tools in existing cryo-CLEM makes the workflow tedious and time consuming – requiring additional training and yielding lower success rate. This proposal aims to develop a cryo-TEM transfer holder with a modular correlative tip containing cryo-sample that allows a single workflow where the sample can be transferred between the vitrification tool, FIB, LM and the TEM. The developed holder tip will have a built-in cryo shutter to prevent the sample from frosting. The correlative tip will interface across all cryo-CLEM tools in the workflow to allow undistorted transfer of sample for high resolution TEM imaging. The developed transfer holder and workflow will be validated onsite at Hummingbird facility as well as using real-world examples with our collaborators – for example looking at host-pathogen interactions. Research groups focused on soft/biological materials interested in accurate and high-resolution analyses of the macromolecular structures are the main customer of this product.Project Narrative Project Title: Cryo-correlative light and electron microscopy (cryo-CLEM) transfer holder for high-resolution imaging Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Khim Karki, Ph.D. Narrative: The cross-correlative electron and optical microscopy characterization techniques that will be developed in this project will have a direct impact on the understanding of biological processes. This project targets correlative high-resolution imaging of biological structures in different microscopes without sample transfer artefacts. This new approach, will be used to gain a comprehensive understanding of cellular structures and function.