Development of a Charge-Resistant Embedding Media for High-Performance Serial Block Face Imaging of Cells and Tissues

Area of Science:

• Electron Microscopy
• Materials Science
• Biotechnology

Background:

• Serial block-face scanning electron microscopy (SBEM) offers high-resolution 3D imaging but is hindered by specimen charging.
• Existing solutions like heavy metal staining or variable-pressure SEM have limitations in resolution or effectiveness.

Purpose of the Study:

• To develop a novel embedding medium that minimizes specimen charging in SBEM without sacrificing imaging quality.
• To investigate the efficacy of polyethylene glycol (PEG 3350) as a doping agent in epoxy resins for improved charge resistance.

Main Methods:

• Incorporating polyethylene glycol (PEG 3350) into standard Durcupan epoxy resin at a 10% concentration.
• Testing the doped resin's performance in high vacuum using charge-prone samples (HeLa cells, lung, brain tissue) at varying keV levels.
• Evaluating specimen integrity, transparency, sectionability, and imaging quality (signal-to-noise ratio, spatial resolution).

Main Results:

• 10% PEG 3350-doped resin substantially reduced charging in diverse biological samples between 1.4-1.8 keV.
• The doped resin maintained sample transparency, sectionability, and high-resolution imaging capabilities in high vacuum.
• Charging was mitigated at voltages (above 1.0 keV) where undoped resins typically show artifacts, with near-complete mitigation achieved at slightly lower voltages for challenging samples.

Conclusions:

• PEG-doped epoxy resins offer a promising solution for minimizing specimen charging in SBEM.
• This approach enables high-quality 3D imaging of sensitive biological specimens.
• Further optimization of PEG concentration and molecular weight could lead to universally charge-resistant embedding media for advanced microscopy.