MicroCT protocols for scanning of embryos and juvenile Hexaplex trunculus
Eva Chatzinikolaou, Kleoniki Keklikoglou
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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.
Steps
Sample preparation
Removal of embryos of Hexaplex trunculus from their egg capsules.
Anesthetization of embryos and juvenile gastropods with 7% MgCl2.
Sample placement inside a plastic white pipette tip without any scanning medium (in the air).
microCT scanning
Equipment
| Value | Label |
|---|---|
| SkyScan 1172 | NAME |
| micro-computed tomographer (microCT) | TYPE |
| Bruker | BRAND |
| 10L01170 | SKU |
| 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 |
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
Images reconstruction
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium)
Upload projection images
Perform X-Y alignment
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
