Preparing Lamellae from Vitreous Biological Samples using a Dual-Beam Scanning Electron Microscope for Cryo-Electron...

Area of Science:

• Structural Biology
• Cell Biology
• Microscopy Techniques

Background:

• Cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET) require high-quality, thin samples for high-resolution imaging.
• Preparing biological samples, such as Plasmodium falciparum-infected human erythrocytes, for cryoEM/cryoET presents challenges in achieving uniformly thin lamellae.
• Robust and contamination-free sample preparation is critical for downstream analyses like sub-tomogram averaging.

Purpose of the Study:

• To present a detailed protocol for preparing cryo-lamellae from plunge-frozen biological samples on-grid.
• To provide a general guide for on-grid cryo-lamella preparation adaptable for various biological specimens.
• To optimize the milling process for achieving uniformly thin lamellae suitable for cryoEM and cryoET.

Main Methods:

• Plunge-freezing of cells on electron microscopy grids into liquid ethane.
• Screening of frozen grids using a light microscope with a cryo-stage.
• On-grid focused ion beam (FIB) milling within a cryo-scanning electron microscope (cryoFIB-SEM) with protective platinum coating applied via gas injection system (GIS).
• Step-wise milling with decreasing ion beam current to achieve electron transparency and minimize heat damage.
• Transfer of milled grids to a transmission electron microscope (TEM) for tilt-series acquisition under cryogenic conditions.

Main Results:

• Successful preparation of cryo-lamellae from Plasmodium falciparum-infected human erythrocytes.
• Demonstration of a robust and contamination-free workflow for on-grid lamella preparation.
• Achieved uniformly thin lamellae critical for high-resolution cryoEM and cryoET imaging.

Conclusions:

• The presented protocol offers a reliable method for on-grid cryo-lamella preparation.
• The protocol is adaptable for diverse biological samples, facilitating cryoEM and cryoET studies.
• Advancements in cryo-lamella preparation are crucial for the progression of cellular cryoEM, cryoET, and sub-tomogram averaging techniques.