MicroCT protocols for scanning and 3D analysis of adult 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 and 3D analysis for the marine gastropod species Hexaplex trunculus.
Steps
Sample preparation
Anesthetization of gastropods with 7% MgCl2
Storage in -20°C
Sample placement inside a custom-made felisol holder 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 adult Hexaplex trunculus
Voltage: 100 kV
Current :100 μA
Filter: aluminium and copper
Pixel size: 13.79 μm
Camera binning: 2 × 2
Exposure time: 2480 ms
Rotation: 180°
Rotation step: 0.60º
Frame averaging: no
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 - 0.13
Save as: 16-bit TIFF images
Image analysis
3D Analysis using the software CT Analyser v.1.18.4.0 (CTAn, Bruker, Kontich, Belgium)
Upload reconstructed images
Creation of a Region of Interest (ROI)
Binary thresholding with a grayscale histogram range 30 - 255 for measuring relative shell density
Custom processing plugin
a) Thresholding: 30-255
b) Despeckle: remove black speckles
c) Despeckle: sweep all except the largest objects
3D Analysis
a) Structure thickness: average value of the diameters of the largest spheres which can be fitted in each point of the shell structure (“sphere-fitting” method)
b) Closed shell porosity: total number of closed pores and % of the closed porosity of the shell (i.e. total volume of enclosed pores of each specimen as a % percentage of the total shell volume)
Save colour coded images for visualisation of the shell structure thichness using the CTVox software (Bruker, Kontich, Belgium)
