Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

The overall goal of this procedure is to prepare a sample of extracellular matrix protein fiber, collagen, or elastin to be mounted in the sample compartment for Brillouin spectroscopy measurements. Using this method, collagen and elastin fibers of the extracellular matrix can be obtained from connective tissues of animal origin for their use in Brillouin spectroscopy measurements. The main advantage of this approach is that the micro-mechanical properties of the sample can be completely determined without prior knowledge of the refractive index of the material.

While the tail is still frozen cut a 20 millimeter long segment from the proximal end and thaw the tissue in a petri dish filled with room temperature PBS. When the tail is thawed, use a scalpel to make an incision along the length of the sample to split the skin, peeling back the tissue to reveal four sheath tendon bundles around the tail vertebrae. Using fine forceps, gently draw each fiber out of the sheath and place them in a vial containing distilled water supplemented with 0.01%weight per volume sodium azide for storage at four degrees Celsius.

To obtain pure fibrous type one collagen immerse the fibers in chondroitinase ABC in tris buffer and sodium acetate for 24 hours at 37 degrees Celsius in a shaking incubator. The next day, transfer the fibers into streptomyces hyaluronidase in tris buffer and sodium chloride for another 24 hours at 37 degrees Celsius in the shaking incubator. Then immerse the fibers in trypsin in sodium phosphate and sodium chloride for 16 hours at 37 degrees Celsius with shaking.

The purified fibers can then be stored at four degrees Celsius in vials of distilled water supplemented with 0.01%sodium azide until needed. To extract bovine nuchal ligament elastin fibers first de-fat the freshly obtained bovine nuchal ligament. When all of the adipose has been removed add the ligament to a conical flask and cover the tissue in freshly prepared sodium hydroxide in distilled water.

Boil the ligament in a 95 degree Celsius water bath for 45 minutes. Then wash the insoluble block repeatedly in a beaker filled with distilled water. When a pH of 7.0 is obtained submerge the tissue in distilled water supplemented with 0.01%sodium azide and seal the container for storage at four degrees Celsius until needed.

After storage, use tweezers to gently pull approximately two millimeter thick, 20 to 50 millimeter long smaller elastin segments from the larger block. Place the segments into a petri dish containing PBS and use fine forceps to gently tease approximately one millimeter thick fiber bundles from the segments. Then use a scalpel to cut the bundles into a few millimeter long fragments and transfer the fiber pieces into vials of distilled water supplemented with 0.01%sodium azide for storage at four degrees Celsius until needed.

To mount the fiber on a reflective substrate first use a diamond cutter to section a piece of reflective silicon slide. Next cut a strip of para-film with a hollow in the center large enough to hold a piece of fiber and place the para-film onto the silicon substrate. Now use a pair of fine forceps to transfer a single fiber from the four degree Celsius storage solution into a small petri dish filled with pure room temperature water.

After 5 minutes, transfer the rinsed fiber into the center of the para-film hollow on the silicon substrate. Then place a thin glass cover slip over the fiber and place a heated soldering iron gently over the glass to melt the para-film beneath the glass. To enable in plane rotation of the sample while maintaining a constant scattering angle and volume position, mount the sample onto a vertical holder equipped with a goniometer and position at 45 degrees to the incident laser beam.

In dry collagen fibers at a specific angle of rotation of zero degrees, longitudinal modes give rise to a bulk peak at 18.92 gigahertz, while the parallel to surface modes give a peak at 9.85 gigahertz. The parallel to surface peak shifts to lower frequencies at the specific angle of rotation approaches 90 degrees, whereas the bulk peak only slightly red shifts upon changing the specific angle of rotation in the same range. In wet collagen fibers the two peaks remain essentially unchanged throughout the experiment with the bulk peak at 10.5 gigahertz and the parallel to surface peak at 4.9 gigahertz indicating an 80 to 100%reduction in the frequency and accounting for the reduced stiffness of the hydrated material.

In this figure a plot of the acoustic wave velocity of dry collagen obtained from parallel to surface and transverse peaks as a function of the specific angle of rotation is shown. In dry elastin fibers measured at a specific angle of rotation of zero degrees the bulk peak occurs at 16.8 gigahertz and the parallel to surface mode occurs at 8.2 gigahertz. The wet elastin fibers however present a bulk peak at a 37%lower frequency than the bulk peak of the dry elastin indicating a reduction in the fiber's stiffness.

As in dry collagen there is evidence of anisotropy in the mechanical properties of dry elastin fibers demonstrating the effectiveness of Brillouin spectroscopy in obtaining relevant data on the stiffness, composition, and structural aspects of specific materials of interest. After watching this video you should have a good understanding of how to prepare collagen and elastin fiber samples for micro-mechanical testing based on Brillouin light scattering spectroscopy.