Micro-CT scanning

EcoEvoDevoLab

Updated 20 December 2024

Image from microphotonics.com

Colby's micro-CT scanner is a Bruker SkyScan 1173. Manuals for its use are available on its accompanying desktop computer. Start by reading Method Note MCT-052 How to Set-up a Scan

A typical scan will create about 25 Gb of data. So be sure the scanner's desktop has that space, and that you have storage space somewhere too!

Procedures

Sample prep

• Stain the specimens. Check the staining protocol intended to visualize thoracic muscle in true bugs. For specimens in ethanol, be sure the stain (e.g. iodone) is not leaking from the sample.
• Immobilize the specimen by packing it in glass wool or tissue paper. – Specimens can be stored in glass or plastic vials. Drinking straws are particualrly good for dried specimens, because they are thin-walled and protect the specimen in storage before and after the scan.
• Various holders are available for different tubes and straws.

Starting a scan

• Launch SkyScan 1173 on the Windows desktop computer. This will start up the machine, which can only be operated remotely.
• Press the Open button to open the main door.
• Clean the chamber using a swiffer duster.
• Secure the sample in the chamber by tighening the metal fixture below the sample holder.
• Press the Close button to close the main door.
• Press the Radiation button to turn on the X-ray source. – If the instrument has been used recently, it should be ready in about 15 minutes. However, if it hasn't been used in a while, it can take several hours to be ready.
• Press the Grab Image button to start a live view or the Camera button for a still image.
• Move the platform down so that the specimen is not in view. Typically a vertical position of 90 mm will do it.
• Update the flat-field correction if the average brightness is not very close to 90%. To do so, Under Preferences, turn off flat-field correction. Then under Scanner Setup for update the black & white flat-field values. This will take a few minutes. Then turn flat-field correction back on. At this point the average should be 90%.
• Position the specimen. On the bar at the bottom right of the window, click the numbers (not the slider) to adjust the position values. These features can also be accessed from the menu Options/Positioning Stage. Try to get the area of interest to fill the frame, but you must leave from blank (flat) space to the left and right. Also beware that the specimen does not colide with the left wall of the chamber! Ballpark starting values:
  • Vertical position: 35 mm
  • Pixel size: 8 um/px (a proxy for distance between the sample and the aperture)
• Center the specimen. Adjust left and right to center the specimen on the green indicator line.
• Rotate the specimen in 90˚ increments and repeat the positioning and centering as needed.
• Press the Start Acquisition button (the circular arrow) to set parameters for the scan.
  • Filename prefix must end in an underscore (_)
  • Make a new folder for each scan under the lab's main folder E:/Results/AngeliniLab/
  • The Rotation turn step parameter will determine the resolution of the scan as well as the time it takes. A scan with 0.1˚ steps will take about 8 hours; 0.2˚ steps will take just under 4 hours.
  • Keep other parameters to defaults, at least initally:
    • Averaging (frames): 3
    • Random movement: do not check
    • 360˚ scanning: check
    • X-ray off after scanning: check
    • Open door: do not check
    • Start reconstruction: do not check

Reconstruction

• Launch GPURecon. This software will run in the background.
• Launch NRecon, the software that will actually perform reconstruction.
• Open any of the images in the scan folder.
• Position the green line for a Z-slice preview, ideally something informative near the center.
• Press Preview in the Reconstruction tab in the upper right.
• Check Use ROI to set a bounding shape, such as an ellipse.
• Adjust histograms (optional).
• Click the Start tab to return to the initial XY side-on view.
• Set the top and bottom bounds for reconstruction. Repeat the preview at levels near the top and bottom to be sure the bounding shape contains the object of interest and not the container, at all levels.
• Click the Settings tab. It can be useful to repeat the preview as you adjust settings to to refine ring reduction and beam hardness corrections. Suggested parameters:
  • post-alignment: 0.5 step
  • beam-hardening correction: 30%
  • ring-artifact reduction: 5 steps
  • smoothing: 1 step
• Click Start. This may take 15-25 minutes depending on the data size of the scan.

Visualization

DataViewer generates 2D combinatorial plots along sagittal, transverse and coronal planes in 3D.

• Launch DataViewer.
• Open an image from the reconstruction folder.
• Click Load for 3D Viewing button on the toolbar.
• Adjust the sagittal, transverse and coronal planes to view features of interest.
• Save the image.

CTVox will create a virtual 3D rendering based on the .

• Launch CTvox.
• Open an image from the reconstruction folder.
• Adjust the histogram levels to define tissues or other materials of interest and to reduce the background.
• Optionally, save the transfer function.
• Move the specimen within the view with the mouse buttons. Hold the left mouse button to move the specimen; hold the right button to rotate it.
• Adjust the orientation of the bounding box by holding CTRL and using the mouse buttons in the same way.
• Adjust the position of the bounding walls by hovering the mouse on them and pressing SHIFT.
• Optionally, add another shape to clip or cut the reconstruction.
• Save the image (or video from the flight interface).

Analysis

• Launch CTAn.
• Open an image from the reconstruction folder.
• Tabs in the right-hand panel define the workflow.
• Use the Region of Interest tab to define a particular structure. This can be repeated by stepping through multiple frames from the top to the bottom of the structure. The software will interpolate.
• Use the Binary Selection tab to adjust histograms to highlight the structure.
• The Morphometry tab will provide some measurements of the structure, such as total area.
• Use the Custom Processing tab to define a 3D surface based on the ROI and binary selection inputs.

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EcoEvoDevo Lab