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Micro-CT Imaging of a Mouse Spinal Cord

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A micro-CT scan is a three-dimensional image that is created from a series of X-ray images at different orientations. The first Nobel prize in physics was awarded to Dr. Rontgen in 1901 for the discovery of X-rays and their use as demonstrated by imaging his wife's hand.

X-ray absorption imaging continues to be an indispensable tool, especially in university labs and hospitals. One of the most advanced uses involves taking a series of two-dimensional X-ray projections to reconstruct a three-dimensional volume. This is known as computed tomography or CT. Micro-CT uses the same basic method, but produces much higher resolution images of smaller volumes.

This video will demonstrate how to obtain X-ray images and use them to produce a micro-CT scan, illustrate the principles of the technology, and finally, discuss some of its applications.

Now let's look at how X-ray images are formed and examine the principles behind assembling them into a micro-CT scan.

A typical micro-CT system features three primary components, an X-ray source, a rotational stage for the sample, and a detector. In the X-ray source, negatively charged electrons are shot in a vacuum where they strike and interact with a target. The electrons decelerate through the target material and emit X-rays. This phenomenon of X-ray generation is known as bremsstrahlung, or braking radiation. The X-rays then leave the source and are either absorbed, scattered, or transmitted by the sample before arriving at the detector. Absorption is the predominant interaction measured in micro-CT, which is due to the large variation in X-ray absorption by different materials in the sample.

Bones contain a lot of atomic calcium and absorb X-rays more so than soft tissue. The absorbed X-rays do not reach the detector and the bones appear white in an X-ray. The output of the tomography is a series of 2D projections at different orientations that can be reconstructed into a 3D volume. The X-ray energy needs to be balanced so there is sufficient attenuation in the sample and signal at the detector.

The intensity, or number of X-rays measured, I, depends on the intensity before attenuation, I-naught, the mass absorption coefficient of the material, mu, the density of the material, rho, and the X-ray path length, X. Ideally, the transmission value I over I-naught, should be between five and 95% for all orientations of the sample, with the best results at the middle range. This value is checked by taking an image of the sample and then dividing the image's pixel values by those in an image of air.

Now that you understand the principles behind micro-CT scans, let's now demonstrate how to produce one.

In this demonstration, a micro-CT scan of the spinal column of a mouse will be obtained.

First, obtain a sample that is suspended in agarose gel. The sample should be cured in a thin walled plastic tube to prevent sample movement and dehydration. The tube walls should be as thin as possible to reduce signal throughput and improve overall image quality.

Then, mount the tube on the sample stage using tape or by making a custom stand. Ensure that the sample is stationary and stable when the stage rotates. Now, turn on the X-ray source and set it to an energy of 90 kiloelectron volts or a voltage of 90 kilovolts, and set the power to eight watts. Once the source warms up, acquire an image through the system software. For automatic acquisition and application, ensure the sample can move in a given direction without crashing. Check the transmission value by normalizing the image against an image of air.

If an image has too high of a transmission, lower the energy incrementally until the transmission value is sufficient. If the image has too low of a transmission, increase the energy incrementally until the transmission value is sufficient. If the sample appears noisy or grainy, increase the exposure time as needed.

Next, move the X-ray source as close to the sample as possible to maximize throughput and obtain the best possible resolution. Be careful not to crash them together. Refine the field of view of the sample by shifting the sample stage using its linear actuators. Then, locate the pixel size of the image. If the CT system supports optical magnification by converting the X-ray signal to visible light signal, try different optical objectives and detector positions. However, be aware that this will affect the scan parameters.

After making necessary adjustments, find the optimal exposure time. Slowly rotate the sample in two-degree increments while monitoring its position relative to the source and detector via the in cabinet camera. Move the source and detector further apart if a collision might occur.

Finally, find the longest X-ray path length that results in the lowest number of counts and determine the exposure time needed for approximately 5,000 counts everywhere.

Now, let's see how a series of images can be acquired. First, select a scan over either 180 degrees or 360 degrees based on the aspect ratio of the sample. For high aspect ratios, select a 180 degree scan, and for low aspect ratios, select a 360 degree scan. If the X-ray path length is four or more times greater in one direction than the other, choose a 180 degree scan.

Next, choose the number of projections and total angular displacement that will dictate the angle between projections. A smaller angle decreases the amount of interpolation of fine feature information, but increases the scan time. A rule of thumb is to have at least 800 projections, but usually no more than 3,200 projections over a 360 degree scan.

Now, submit the scan. The full series of X-ray images will take on the order of a few to tens of hours to acquire. Once the scan is complete, load the series of 2D images into the reconstruction software. Now, select the optimal center shift corrections so that the images line up around a shared axis. This value is usually somewhere between negative ten and ten pixels.

Next, select the optimal beam hardening correction coefficient. This removes false contrast deriving from low energy X-ray attenuation. An average value is somewhere between zeri and 0.5. Then submit the reconstruction. Once the micro-CT scan has been reconstructed, the results are ready for analysis.

Here is a representative micro-CT scan that was obtained using this procedure. Here, we see the 3D volume of a mouse spinal cord. Further image processing two digital cross-sectional slices allows quantitative data such as material porosity, and feature size can be obtained using software tools. The spacing between the sections of a vertebrae and intervertebrae passageways was measured to be on the order of hundreds of microns.

Here is another micro-CT scan that was obtained of a rat's knee. We can see the porosity of the cortical bone and can measure the spacing within the cortical bone of a rat's knee and the thickness of the articular cartilage.

You have just seen a micro-CT scan of a mineralized biological sample, but the applications of 3D X-ray tomography extend to the worlds of microelectronics, geology, additive manufacturing, fuel cells, and more. We will examine a few other instances.

High resolution X-ray images of animal soft tissues can be obtained despite their natural low X-ray absorption. This is accomplished by use of simple contrast staining. In this example, a mouse hind brain is stained using Lugol's iodine solution prior to imaging. The sample is then prepared, loaded, and X-ray images are taken. Finally, a micro-CT scan is created clearly showing lesions in the hind brain.

Micro-CT can be used to characterize the micro structure of electronic devices. In this example, an LED is scanned. Micro-CT scans enable engineers to analyze device failure or reverse engineer a device.

Three-dimensional structures can be created from micro-CT data. In this example, a rat is anesthetized and scanned. The data can then be analyzed to distinguish bone structure from surrounding tissue. Finally, a physical model of the result can be created using a 3D printer.

You've just watched JoVE's introduction to creating 3D micro-CT scans from 2D X-ray images. You should now understand the principles behind X-ray imaging, the relationship between X-ray images and CT scans, how to produce a micro-CT scan of a sample, and some applications. Thanks for watching!

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