Sep 03, 2021
MicroCT protocols for scanning of embryos and juvenile Hexaplex trunculus
- 1Hellenic Centre for Marine Research
Protocol Citation: Eva Chatzinikolaou, Kleoniki Keklikoglou 2021. MicroCT protocols for scanning of embryos and juvenile Hexaplex trunculus. protocols.io https://dx.doi.org/10.17504/protocols.io.bxw4ppgw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 02, 2021
Last Modified: September 03, 2021
Protocol Integer ID: 52924
Keywords: microCT, gastropods, embryos, juveniles, scanning, Hexaplex trunculus
Funders Acknowledgement:
ECCO project, Hellenic Foundation for Research and Innovation ( HFRI)
Grant ID: project ID 343
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Abstract
Micro-computed tomography (micro-CT) is a high-resolution 3D-imaging technique which is now increasingly applied in biological studies focusing on taxonomy and functional morphology. The creation of virtual representations of specimens can increase availability of otherwise underexploited and inaccessible samples. This protocol aims to standardise micro-CT scanning procedures for embryos and juveniles of the marine gastropod species Hexaplex trunculus.
Sample preparation
Sample preparation
42m
Removal of embryos of Hexaplex trunculus from their egg capsules.
10m
Anesthetization of embryos and juvenile gastropods with 7% MgCl2.
30m
Sample placement inside a plastic white pipette tip without any scanning medium (in the air).
2m
microCT scanning
microCT scanning
2h 23m
Equipment
SkyScan 1172
NAME
micro-computed tomographer (microCT)
TYPE
Bruker
BRAND
10L01170
SKU
LINK
Detail detectability: <1 μm
Low contrast resolution (10% MTF): 5 μm
Pixel size at maximum magnification: <0.8 μm
X-ray source:
Sealed microfocus X-ray tube, air cooled, >10,000h lifetime;
Spot size <5μm @ 4W, 20-100kV, 0-250 μA (10W max)
X-ray detector (camera): 11 Megapixel (4000X2300) 12-bit digital CCD-camera with fibre optic coupling to scintillator
Maximum object size: 50 mm in diameter using offset scan
Radiation safety: < 1 µSv/h at any point on the instrument surface
SPECIFICATIONS
2h 23m
Scanning parameters for embryos and juvenile Hexaplex trunculus
Voltage: 59 kV
Current :167 μA
Filter: none
Pixel size: 2 μm
Camera binning: 1 × 1
Exposure time: 325 ms for embryos and 316 ms for juveniles
Rotation: 360° for embryos and 180° for juveniles
Rotation step: 0.20º for embryos and 0.25° for juveniles
Frame averaging: 3 for embryos and 5 for juveniles
2h 23m
Images reconstruction
Images reconstruction
20m
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium)
20m
Upload projection images
2m
Perform X-Y alignment
2m
Reconstruction parameters
Smoothing: 2
Ring artifact correction: 20
Beam hardening correction: 59%
Attenuation coefficients: 0 - 1.127 for embryos and 0 - 1 for juveniles
Save as: 16-bit TIFF images
16m