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

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.The relevant structures in biological specimens span over a wide range of length scales 1-3 . To obtain a holistic understanding of such structures, correlative microscopy combines multiple imaging techniques to investigate a single sample. It thereby exploits the specific strengths of the different imaging modalities, particularly with respect to sample sizes and resolution, to answer modern life science questions 1,2,4 .Among microscopic techniques correlative light and electron microscopy (CLEM) has been found to be particularly powerful. Both imaging fields are highly complementary 5 . CLEM is a collective term for all different combinations of light and electron microscopy, ranging from fluorescence super-resolution microscopy to conventional bright field light microscopy and from focused-ion-beam scanning electron microscopy (FIB-SEM) over serial-block-face SEM (SBF-SEM) to transmission electron microscopy (TEM) 1,4-6 . 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 .X-ray computed tomography (CT) is a widely used tool for non-destructive 3D imaging with isotropic resolution 3,9,10 . Recently, high-resolution X-ray CT (terminology used in this study: microCT resolutions around 1 µm, NanoCT resolutions around 500 nm and smaller) has been integrated in a growing number of correlative studies 1,7,[11][12][13] .Combining different i...

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Methods to study organogenesis in decapod crustacean larvae II: analysing cells and tissues

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Correlative Microscopy: a tool for understanding soil weathering in modern analogues of early terrestrial biospheres

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