EVOH Hollow Fiber Encapsulation of Individual Caenorhabditis elegans for Transmission Electron Microscopy

Abstract

We present a streamlined protocol for encapsulating individual Caenorhabditis elegans specimens for thin-section transmission electron microscopy (TEM) using ethylene vinyl alcohol (EVOH) copolymer hollow fibers. These transparent fibers, featuring a 175 m inner diameter, 25 m wall thickness, and an approximate molecular weight cutoff of 30 kDa, possess both hydrophilic and hydrophobic properties. This amphiphilic nature helps maintain a potent lumen structure even in dry, open air, enabling precise handling and orientation of small specimens prior to resin polymerization and eliminating the need for remounting. The protocol includes specimen transfer using wire loops, microwave-assisted chemical pre-fixation, dissection on a soft silicone substrate with micro-scissors, and manipulation of hollow fibers using insect pins. This approach preserves the identity of individual specimens throughout processing. During ultramicrotomy, the fiber serves as a visual guide, enhancing alignment and reproducibility of sectioning. As proof of principle, we demonstrate well-preserved ultrastructural features in chemically fixed, microwave- assisted resin-embedded C. elegans specimens. This method offers a robust and reproducible alternative to conventional gelatinous media encapsulation, particularly suited for TEM analysis of scarce or small biological samples.