High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

The overall goal of this procedure is to perform 3D x-ray micro tomography of mineralized and non mineralized tissues. This is accomplished by first extracting the samples to be examined, then immobilize and position the samples in the micro CT instrument. The third step of the procedure is to set acquisition parameters for each sample and collect the images.

Ultimately, a 3D image of the sample at high resolution through x-ray CT is obtained. The main advantage of this technique of our existing methodologies, like MRI, ultrasound or electron microscopy, is that it provides information about tick volumes at one micron resolution D.This method can provide insight in many biological tissues. It can be used also in electronics, in the material sciences, and in archeology.

Preparation is different for mineralized and non mineralized tissue. Mineralized tissues must be sealed in containers with a tight fit, so sample position does not change. While high resolution measurements are made, the container shape will vary according to the tissue's morphology.

For example, a mouse femur extracted from an 18.5 D old embryo can fit into a pipette tip. Begin by sealing the narrow end of a polystyrene pipette tip with glue like epoxy resin. Then fill the tip with a working buffer.

In this case, PBS now fit the leg with exposed femur tightly into the tip and place the pipette tip into a suitable holder and seal the other end with para film sheet sacrifice and perfuse the animal as outlined in the accompanying manuscript. Then extract the stained lungs and heart and transfer them to a prepared 50 milliliter test tube. Place the organs on a dry piece of paper.

Place the samples in the final measuring test tubes at the bottom of the test tube is an ethanol dampen cloth. To create an ethanol saturated environment, the sample should fit tightly within the tube. If the sample is loose, tighten it with a wire and immobilize it near the bottom of the tube just above the cloth.

Next, glue or screw the tube into a holder of the instrument and proceed with setting the image. Acquisition parameters. Begin by placing the sample holder in the rotation stage of the instrument.

Then using arbitrarily selected values for voltage and current, take an x-ray image. If the image is too dark. Make changes by first gradually increasing the number of photons.

This is done by progressively increasing the current. If the image hasn't gotten brighter after increasing the current to 200 microamps, then make small progressive increases to the energy of the x-ray photons by increasing the voltage. If the image is too bright, first, decrease the voltage, then decrease the current until the image is satisfactory.

Benning can also be used to increase image brightness at the sacrifice of resolution. A inning value of two will make the image about four times brighter at half the resolution. After setting the optimum brightness, optimize the exposure time of the camera to compromise between the best obtainable contrast with a reasonable experiment duration.

Image contrast, especially for samples with high absorption, can be improved by using filters to reduce low energy photon flux. Now, choose the working magnification between 0.5 x and 40 x and fit the field of view to encompass the whole sample while maximizing resolution. To increase the resolution, place the x-ray source closer to the sample.

To increase the field of view, place the detector closer to the sample. For 3D imaging, the sample must fit within the field of view at any angle of rotation, so the axis of rotation must be centered. Begin by rotating the sample to negative 20 degrees.

If the desired volume has shifted laterally, reposition the rotation axis. Proceed with taking images at further rotations and continue making corrections until the sample is fully visible from negative 90 to positive 90 degrees, then proceed with measurement at all angles between negative 90 and 90 degrees. Running an imaging program can take a long time, so plan accordingly.

For example, to image the most femur at Forex magnification with eight micron resolution requires 1000 projection images. This only takes three hours to complete. However, it takes 10 hours to collect the 2, 500 projection images needed to visualize rat lungs at 0.5 x magnification and 16 micron resolution.

For future image comparison, it is necessary to calibrate the image by taking images of a standard phantom made of artificial materials that have bone like and waterlike x-ray absorption under the same experimental conditions. As for the sample, after recording all projection images, the full volume is reconstructed. To calibrate the reconstructed image to the values for the phantom, use the hounds field or CT scale.

For example, at Forex magnification, the waterlike value in the phantom is set to zero and the bone like value is set to 3000 from this range. All other values are interpolated or extrapolated. The images can now be analyzed using any software package that can support 20 gigabyte files, such as freeware image J, or Fiji.

This volume rendering shows the femur of a mouse four days after bone mineralization has initiated the mineralized fraction calculated at 18%and the bone mineral density can be compared to bones in other stages of development. This tomographic volume rendering of lungs from a 12 week old female nude rat is resolved to 16 microns. Pulmonary staining is used to reveal 20 micron diameter blood vessels.

In this serial section analysis of the same lungs. Multiple cancer nodules can be seen as solid gray shapes within four weeks of implanting tumor cells. In these lungs, nodules grew to cover 17%of the lungs volume.

Most of the pulmonary staining was found in the peripheral areas of the tumors. Blood vessels are also present inside the nodules, covering some 3%of their volume. Once mastered, sample positioning can be done in 15 to 20 minutes if it is performed properly.

The imaging time including automatic volume reconstruction, which usually does not require the presence of an operating person, depends on the sample and may take as long as 50 hours. Don't forget that working with fixating and staining agents can be extremely hazardous. Always wear lab gloves and avoid breathing vapors from the samples.