Enhancing EMSL’s cryo-EM capability through a novel high pressure freezing workflow to improve throughput and sample quality
EMSL Project ID
60839
Abstract
Cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) is an essential sample preparation technique for creating thin sections (500nm or less) of cells and tissues for cryo-transmission electron microscopy (cryo-TEM) that would otherwise be too thick for imaging.(1, 2) Cryopreservation of cells and tissues larger than ~5µm in diameter however requires high pressure freezing (HPF), which results in a sample preparation pathway that considerably lowers the throughput of generated thin sections for downstream cryo-TEM analysis. Since there are many existing EMSL user projects and an increasing number of inquiries from the BER community that target plant tissues or organisms which are larger than this ~5 µm size threshold, there is a need to develop a workflow which allows for cryopreservation of samples compatible with higher throughput through cryo-FIB/SEM on-grid thinning. This proposed workflow fills that need and directly supports the Bioatomic Imaging Roadmap with increased sample throughput. The first objective of this proposal is to establish a workflow to reliably produce so called “waffle grids” where samples are frozen at controlled thicknesses on TEM grids in combination with cryoprotectants to limit the formation of ice crystals using HPF.(3) The second objective of this proposal is to demonstrate the successful vitrification of the sample at controlled thicknesses, using on-grid lamella thinning and cryo-TEM imaging. It is expected that the implementation of this workflow will not only increase the number and types of organisms which may be accessed with EMSL’s cryoEM capability, but also increase throughput of cellular cross sections for imaging by 5x.
Project Details
Start Date
2023-04-17
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Team Members