MicroCT protocols for scanning and 3D analysis of adult Hexaplex trunculus
Sep 02, 2021

MicroCT protocols for scanning and 3D analysis of adult Hexaplex trunculus

  • 1Hellenic Centre for Marine Research
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
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.
 
Eva Chatzinikolaou, Kleoniki Keklikoglou 2021. MicroCT protocols for scanning and 3D analysis of adult Hexaplex trunculus. protocols.iohttps://dx.doi.org/10.17504/protocols.io.bxwqppdw
ECCO project, Hellenic Foundation for Research and Innovation ( HFRI)
Grant ID: project ID 343
microCT, gastropods
 protocol  ,  
Sep 02, 2021
Sep 02, 2021
 
52912
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.

Sample preparation

34m
1
30m
Anesthetization of gastropods with 7% MgCl2
2
2m
Storage in -20°C

3
2m
Sample placement inside a custom-made felisol holder without any scanning medium (in the air)

microCT scanning

5h 20m
4

SkyScan 1172
micro-computed tomographer (microCT)
Bruker
10L01170
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


4.1
5h 20m
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

3h
5
3h
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium)

5.1
Upload projection images
5.2
Perform X-Y alignment
5.3
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

5h 37m
6
3D Analysis using the software CT Analyser v.1.18.4.0 (CTAn, Bruker, Kontich, Belgium)


6.1
10m
Upload reconstructed images
6.2
5m
Creation of a Region of Interest (ROI)
6.3
30m
Binary thresholding with a grayscale histogram range 30 - 255 for measuring relative shell density
6.4
1h 10m
Custom processing plugin

a) Thresholding: 30-255
b) Despeckle: remove black speckles
c) Despeckle: sweep all except the largest objects
6.5
3h 40m
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)
6.6
2m
Save colour coded images for visualisation of the shell structure thichness using the CTVox software (Bruker, Kontich, Belgium)