Cryo-FIB-lift-out: practically impossible to practical reality.

Area of Science:

• Materials Science
• Biophysics
• Microscopy

Background:

• Sample preparation for cryogenic transmission electron microscopy (cryo-TEM) is challenging for hydrated biological and soft-matter materials.
• Traditional (in situ) Lift-Out is effective for room temperature samples but not directly applicable to cryo-preserved specimens.

Purpose of the Study:

• To detail the development and practical application of the Cryo-Lift-Out (cryo-LO) technique for cryo-TEM sample preparation.
• To provide a comprehensive guide for researchers new to cryo-LO, covering hardware, principles, and protocols.
• To highlight current achievements and future directions for cryo-LO technique development.

Main Methods:

• Development and refinement of the cryogenic Lift-Out (cryo-LO) technique.
• Focused Ion Beam (FIB) milling to create lamellar samples.
• Detailed protocol development for cryo-LO sample preparation.

Main Results:

• Cryo-LO has evolved from a theoretical concept to a state-of-the-art practical reality for sample preparation.
• Established key hardware, basic principles, and practical considerations for implementing cryo-LO.
• Presented detailed protocols and identified areas for further development.

Conclusions:

• The Cryo-Lift-Out technique is a viable and advanced method for preparing high-quality samples for cryo-TEM.
• This work formalizes existing knowledge and provides a foundational resource for new users.
• Continued development promises further advancements in cryo-TEM sample preparation.