Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

We implement microCT with phosphotungstic acid to enhance the soft tissue contrast in the non-invasive investigation of the human orbicularis retaining ligament, a delicate structure within the orbit area. This protocol has been optimized to aid researchers in the non-invasive investigation of human soft tissues and is simple to perform with a reasonable amount of expense. To obtain the samples, first use a colored pencil to indicate the cutting area on the skin for the sample harvesting.

Confirm that the marked line extends medially to a medial campus, laterally to a lateral campus, superiorly to a superior border of the lower eyelid, and inferiorly to one centimeter below the line from the orbital rim. Next, use a blade to cut the facial tissues along the marked line, making sure that the cut is deep enough for the blade tip to touch the bone. The harvested sample must include the skin, subcutaneous tissue, muscle, fat, and periosteum.

Upon collection, immediately fix the sample in 10%formalin for five to seven days at room temperature. At the end of the fixation period, slice the sample in three five to seven-millimeter thick pieces. Use a needle and black thread to sew the superolateral side of each sample such that the direction of the sample can be determined at later time points.

Dehydrate the sample in an ascending ethanol concentration series for one day per concentration, leaving the samples in the 70%ethanol solution until they're staining. One week before the microcomputer tomography scanning is scheduled, add 2.1 grams of PTA to 210 milliliters of 70%ethanol and place the solution on a shaker at 55 to 60 rotations per minute. Prepare three 70-milliliter plastic containers for each sliced piece.

When the dye has dissolved into the alcohol solution, fill the containers with the solution. Place one specimen into each container of 1%PTA, and shake the samples at 55 to 60 rotations per minute for five to seven days. When the staining is complete, store the samples in fresh 70%ethanol for one week to prepare for the scanning.

For microCT scanning, wrap the samples in Parafilm to prevent drying and place a sample onto the scanner tray. Set the scanner's source voltage to 70 kilovolts, the source current to 114 microamps, the aluminum filter to 0.5 millimeters, the image pixel size to 20 squared micrometers, the pixels to 2, 240 by 2, 240, the exposure to 500 milliseconds, and the rotation step to 0.3 degrees. Then, initiate the scanning.

When the scanning is complete, open the reconstruction software and select Actions and Open Dataset to launch the scanned files. Select the Settings tab and set the ring artifacts reduction to seven and the beam-hardening correction. To begin the reconstruction, select Start.

The final data will be stored in the designated folder. At the end of the reconstruction, open the file resizing software, and select Source data set to launch the reconstructed files. Select jpg on the Destination data set tab and select the 1/2 Resizing option with the Quality option of No interpolation Then adjust the slide bar to 100 in the Image compression tab and start the conversion.

For 3D reconstruction, open the 3D volume rendering software, and select Actions and Load Volume Data. To adjust the brightness and contrast level, modify the shape transfer function in the histogram in the Transfer Function Editor tab. Next select Options and Lighting, and select the Shadows and Surface Lighting icons to achieve a realistic modeling tone.

Click and drag, and right-click and drag to move and rotate the 3D image to find the best view, scrolling to zoom in or out as desired. Click Shift and drag to slide the place to view the sectional images. Turn on the Light icon and adjust the lighting indication bar to find the best lighting for viewing, then turn off the Light icon and close the Lighting tab.

In the Options tab, select Show and Clipping Box to hide the box for the final image. Then, select Actions and Save Image to store the image. This detailed reconstruction of the ORL was achieved by microCT with PTA preparation as demonstrated, allowing visualization of a ligamentous fibromuscular structure extending obliquely between the dermis and the periosteum.

In the coronal view, the amount and complexity of the fibers increases laterally. In horizontal view, an elaborate meshwork with an arborized formation is observed. In the sagittal view, the thicknesses of the ORL fibers decreases inferiorly.

Overall, this multi-directional observation proves that the ORL is made up of a multilayered meshwork of continuous plates with variations in the number and thickness of the fibers depending on their location. When the duration of the staining is insufficient compared to the volume of a specimen, the final image may include an empty hole in the central area of the specimen. Other agents beside PTA can be used and different agents have different merits or staining features interests.

Therefore, it is useful to optimize the staining region according to your experiment.