MicroCT protocols for scanning egg capsules of 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 the egg capsules of the marine gastropod species Hexaplex trunculus.
Steps
Sample preparation
Fixation of egg capsules of Hexaplex trunculus in 5% formaldehyde buffered with seawater.
Wash egg capsules with distilled water and dehydrated them with ethanol in gradually increasing concentrations (20%, 50%, 70%, 96%).
Staining of egg capsules with 1% iodine in 96% ethanol.
Washing of stained samples with 96% ethanol.
Placement of samples inside a plastic Falkon tube with 96% ethanol as a scanning medium.
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: 80 kV
Current :124 μA
Filter: aluminium
Pixel size: 13.79 μm
Camera binning: 2 × 2
Exposure time: 1435 ms
Rotation: 360°
Rotation step: 0.40º
Frame averaging: 3
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 - 0.064
Save as: 16-bit TIFF images
