Nanoscopic X-ray tomography for correlative microscopy of a small meiofaunal sea-cucumber

Simone Ferstl,Mark Müller,Martin Dierolf,Bernhard Ruthensteiner,Sebastian Allner,Klaus Achterhold,Franz Pfeiffer,Lorenz Hehn,Thomas Schwaha

doi:10.1038/s41598-020-60977-5

Abstract

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology. Due to sample thickness limitations, these imaging techniques often require sectioning to investigate small animals and thereby suffer from various artefacts. A recently introduced nanoscopic X-ray computed tomography (NanoCT) setup has been used to image several biological objects, none that were, however, embedded into resin, which is prerequisite for a multitude of correlative applications. In this study, we assess the value of this NanoCT for correlative microscopy. For this purpose, we imaged a resin-embedded, meiofaunal sea cucumber with an approximate length of 1 mm, where microCT would yield only little information about the internal anatomy. The resulting NanoCT data exhibits isotropic 3D resolution, offers deeper insights into the 3D microstructure, and thereby allows for a complete morphological characterization. For comparative purposes, the specimen was sectioned subsequently to evaluate the NanoCT data versus serial sectioning light microscopy (ss-LM). To correct for mechanical instabilities and drift artefacts, we applied an alternative alignment procedure for CT reconstruction. We thereby achieve a level of detail on the subcellular scale comparable to ss-LM images in the sectioning plane.

Highlights

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology
With our in-house-built NanoCT setup, we analyzed a specimen of Leptosynapta cf. minuta which is an only little investigated small representative of a sea cucumber (Fig. 1a, Movie S1), adapted to a meiofaunal lifestyle
The sample was prepared for transmission electron microscopy (TEM) imaging

Summary

Introduction

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology. Among microscopic techniques correlative light and electron microscopy (CLEM) has been found to be powerful Both imaging fields are highly complementary[5]. Since light and electron microscopy techniques conventionally feature limited penetration depths through matter, they typically rely on sectioning or complete destruction of the sample to create 3D information of entire small biological specimens[1,7,8]. This often prevents isotropic resolution, and the resulting data are affected by deficiencies such as sectioning and alignment artefacts[1,4,5]. It is highly relevant to minimize the complexity of sample processing

Methods

Results

Conclusion