Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

Abstract

Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

High-precision targeting workflow for volume electron microscopy

“…We show that FIB-SEM targeting can be achieved with micrometer precision based exclusively on fluorescence, without relying on anatomical or morphological features. Despite the low amount of heavy metals in the sample and the lower hardness of the resin used, the imaging quality enabled fine ultrastructural analysis, at par with traditional protocols (Polilov et al, 2021), demonstrating that FIB-SEM is compatible with a large spectrum of sample preparation procedures.…”

The 3D ultrastructure of the chordotonal organs in the antenna of a microwasp remains complex although simplified

“…Adult female Megaphragma viggianii Fusu, Polaszek and Polilov 2022 (Hymenoptera: Trichogrammatidae) were reared from eggs of Heliothrips haemorrhoidalis (Bouché, 1833) (Thysanoptera: Thripidae). Detailed protocol can be found in previous publication 49 . The head was dissected from the body in the cold fixative and immediately thereafter transferred to fresh fixative of 4 °C for 1 h, which consisted of 1% glutaraldehyde (GA) and 1% osmium tetroxide (OsO 4 ) in 0.1 M sodium cacodylate buffer (pH = 7.2).…”

Porous or non-porous? The challenge of studying unusual placoid sensilla of Megaphragma wasps (Hymenoptera, Trichogrammatidae) with electron microscopy

“…The analysis of internal ultrastructure was based on the three-dimensional electron microscopy data obtained from a custom FIB-SEM (Zeiss Merlin scanning electron microscope with a Zeiss Capella focused ion beam), following fixation of the samples, en block staining, and embedding in EPON (for more details, see Polilov et al (2021)).…”