Automated in-chamber specimen coating for serial block-face electron microscopy.

Area of Science:

• Materials Science
• Microscopy Techniques
• Nanotechnology

Background:

• Insulating specimens in scanning electron microscopy (SEM) suffer from sample charging, distorting images.
• Traditional conductive coating methods are incompatible with serial block-face electron microscopy (SBEM) due to the in-situ cutting and imaging process.

Purpose of the Study:

• To develop a method to eliminate charging effects in SBEM for insulating samples.
• To improve image quality and enable advanced imaging modes in SBEM.

Main Methods:

• Integrated an electron beam evaporator into the SEM chamber for in-situ metallic film coating (1-2 nm) after each cutting cycle.
• Automated the coating process for serial acquisition of SBEM stacks.
• Utilized beam deceleration for enhanced contrast on non-conducting samples.

Main Results:

• Successfully eliminated charging effects for both backscattered (BSE) and secondary electron (SE) imaging on samples up to 12 mm.
• Observed minimal signal-to-noise ratio (SNR) reduction compared to low-vacuum methods.
• Demonstrated acquisition of over 1000 cut/coat/image cycles and successful imaging using beam deceleration and SE contrast.

Conclusions:

• In-situ coating is a viable solution for charging artifacts in SBEM.
• This method preserves sample integrity and enhances imaging capabilities for insulating materials.
• The automated process facilitates high-resolution 3D reconstructions of challenging specimens.