A Practical Approach to Study Uncultivated Protists Using Single-Cell Techniques for Electron Microscopy.

Protists represent a significant portion of eukaryotic diversity with a wide range of ecological roles, lifestyles, and diverse morphological traits. Despite their widespread importance in ecological systems and their potential as model organisms, most protist lineages remain poorly characterized, in part due to their small size and the fact that many lineages remain uncultivated. High-resolution microscopy techniques like transmission and scanning electron microscopy (TEM and SEM) are powerful tools for studying protists, but the application of these techniques has significant limitations when applied to uncultivated species, largely due to the absence of reproducible methodologies tailored to studying single cells with small sample sizes. We present a robust protocol for preparing individual eukaryotic cells for TEM and SEM that addresses these limitations, can be implemented in the field, and uses inexpensive and easily obtainable materials. Our method minimizes cell loss during sample preparation for TEM and SEM, and it enables the tracking of single cells through TEM preparation to ensure cells can be localized and oriented appropriately for ultramicrotome sectioning. This protocol expands the feasibility of ultrastructural studies on uncultivated protist lineages and aims to make high-resolution microscopy more accessible for the broader community of protistologists who study uncultivated taxa.