Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Sci Rep
2020 Mar 03;101:3960. doi: 10.1038/s41598-020-60977-5.
Show Gene links
Show Anatomy links
Nanoscopic X-ray tomography for correlative microscopy of a small meiofaunal sea-cucumber.
Ferstl S
,
Schwaha T
,
Ruthensteiner B
,
Hehn L
,
Allner S
,
Müller M
,
Dierolf M
,
Achterhold K
,
Pfeiffer F
.
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.
Figure 1. Morphology of Leptosynapta cf. minuta. Photograph (a), NanoCT volume renderings (effective voxel size ~540 nm) (b–g). (a) Overview over the alive specimen with an approximate body size of 1 mm. (b) Virtual section through the NanoCT 3D volume rendering exposing the segmented inner organs. (c–g) NanoCT 3D renderings depicting the body and the segmented organs in anterior (c), posterior (d), lateral (e), obliquely dorsal (f), ventral (g) views. Legend: cr: calcareous ring, ec: epidermal cups, g: gut, lm: longitudinal muscle, mo: mouth opening, rc: radial canal in tentacle, ri: ring canal, rn: radial nerve, sc: statocyst, st: stone canal, t: tentacle, ov: ovary. Scalebars: 100 µm.
Figure 2. Co-registration of overview data from ss-LM (10x objective lens) and NanoCT (effective voxel size ~540 nm). Note that the grey values of the histological section images are inverted for better comparison. (a) 3D renderings of ss-LM data (left, blue/green) and NanoCT (right, grey) displayed adjacent to each other. (b) Plane of interest, which is depicted in (c,d), outlined in the volume rendering. (c) Virtual section of the ss-LM after alignment through the plane marked in (b). (d) Corresponding NanoCT slice through the same plane shown in (b). Legend: I: co-registration mismatch, II: alignment artefact, lm: longitudinal muscle, nr: nerve ring, pv: polian vesicle, rn: radial nerve. Scalebars: 100 µm.
Figure 3. Comparison of high-resolution data from ss-LM (20x objective lens) and NanoCT (effective voxel size ~290 nm). Note that the grey values of the histological section images are inverted for better comparison. (a) Planes of interest, which are displayed in (b–g), outlined in the volume rendering. From left to right: I (shown in b,c), II (shown in d,e), III (shown in f,g). (b) ss-LM slice through plane I shown on the left in (a). (c) Corresponding NanoCT slice through plane I. (d) ss-LM slice through plane II displayed in (a). (e) Corresponding NanoCT slice through plane II. (f) ss-LM slice through plane III shown in (a). (g) Corresponding NanoCT slice through plane III. Legend: ec: epidermal cups, g: gut, lm: longitudinal muscle, nc: nucleolus, ne: nervous system, ov: ovary, ri: ring canal, rn: radial nerve, sc: statocyst. Scalebars: 100 µm.
Figure 4. Comparison of the CNR of NanoCT and LM. (a), (b) LM section image and corresponding high-resolution NanoCT slice (effective voxel size ~290 nm) with different ROIs. (c,d) Bar chart of the mean value and the standard deviation of the LM and NanoCT data within the ROIs shown in (a,b). For reference the mean value of resin was set to 0. (e) CNR values with resin as reference of the LM and NanoCT data within the ROIs in (a,b). Scalebars: 100 µm.
Figure 5. Comparison of the spatial grey value profile along two lines in the NanoCT and LM data illustrating the resolution of the two imaging modalities. (a,b) LM section image and corresponding high-resolution NanoCT slice (effective voxel size ~290 nm) with the chosen lines indicated. (c,d) Spatial distribution of the grey values along line 1, line 2 in the LM and NanoCT data. Scalebars: 100 µm.
Figure 6. Comparison of filtered-back-projection (FBP) reconstructed high-resolution NanoCT data (effective voxel size ~290 nm) with either conventional centre shift correction or the novel alignment metric. (a) Centre shift corrected FBP reconstructed slice of the high-resolution NanoCT data. (b) FBP reconstructed slice using the advanced alignment metric of the high-resolution NanoCT data. (c) Detail image of the blue marked ROI in (a). (d) Detail image of the blue marked ROI in (b). Legend: lm: longitudinal muscle, ri: ring canal, rn: radial nerve. Scalebars: 100 µm.
Atwood,
Fine structure of spermatogonia, spermatocytes, and spermatids of the sea cucumbers Cucumaria lubrica and Leptosynapta clarki (Echinodermata: Holothuroidea).
1974, Pubmed,
Echinobase
Atwood,
Fine structure of spermatogonia, spermatocytes, and spermatids of the sea cucumbers Cucumaria lubrica and Leptosynapta clarki (Echinodermata: Holothuroidea).
1974,
Pubmed
,
Echinobase
Bech,
X-ray imaging with the PILATUS 100k detector.
2008,
Pubmed
Burnett,
Correlative tomography.
2014,
Pubmed
Bushong,
X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.
2015,
Pubmed
Caplan,
The power of correlative microscopy: multi-modal, multi-scale, multi-dimensional.
2011,
Pubmed
Giepmans,
Bridging fluorescence microscopy and electron microscopy.
2008,
Pubmed
Gross,
X-ray imaging of a water bear offers a new look at tardigrade internal anatomy.
2019,
Pubmed
Gureyev,
On the "unreasonable" effectiveness of transport of intensity imaging and optical deconvolution.
2017,
Pubmed
Gürsoy,
Rapid alignment of nanotomography data using joint iterative reconstruction and reprojection.
2017,
Pubmed
Handschuh,
A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario.
2013,
Pubmed
Hehn,
Blind deconvolution in model-based iterative reconstruction for CT using a normalized sparsity measure.
2019,
Pubmed
Herdina,
Correlative 3D-imaging of Pipistrellus penis micromorphology: Validating quantitative microCT images with undecalcified serial ground section histomorphology.
2015,
Pubmed
Jahn,
Evaluation of contrasting techniques for X-ray imaging of velvet worms (Onychophora).
2018,
Pubmed
Kraft,
Performance of single-photon-counting PILATUS detector modules.
2009,
Pubmed
Lucas,
Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.
2012,
Pubmed
Mayo,
Software image alignment for X-ray microtomography with submicrometre resolution using a SEM-based X-ray microscope.
2007,
Pubmed
Miller,
Molecular phylogeny of extant Holothuroidea (Echinodermata).
2017,
Pubmed
,
Echinobase
Müller,
Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.
2017,
Pubmed
Paganin,
Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object.
2002,
Pubmed
Parkinson,
Automatic alignment and reconstruction of images for soft X-ray tomography.
2012,
Pubmed
Patel,
Self-calibration of a cone-beam micro-CT system.
2009,
Pubmed
Samei,
A method for measuring the presampled MTF of digital radiographic systems using an edge test device.
1998,
Pubmed
Schneider,
Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging.
2012,
Pubmed
Smith,
Quantitatively imaging chromosomes by correlated cryo-fluorescence and soft x-ray tomographies.
2014,
Pubmed
Sombke,
Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: a methodological and comparative survey.
2015,
Pubmed
Walton,
Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography.
2015,
Pubmed
