Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The overall goal of this procedure is to determine the structure, chemistry, and mechanical properties of multi-layered mineralized fish scales by performing x-ray computed tomography, nano indentation for your transform infrared spectroscopy and scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. This is accomplished by first preparing the individual scales for analysis by removing the soft tissue from the scales, mounting and sectioning the scales, and then performing stepwise polishing. The second step is to perform spatially correlated nano indentation along the cross section of the scale with a diamond percovich tip to determine the local nano mechanical properties.

Next, the inner and outer fish scale layers are examined with an FTIR microscope to identify the main functional groups in the respective layers. The final step is to use the EDX detector on the scanning electron microscope to correlate the local chemical composition to the nano indentations made in the fish scales and to capture images of the indents in the different layers and fracture surfaces. Ultimately, by combining these various experimental techniques, the natural design principles of mineralized fish scales are elucidated through an understanding of the structure property relationships.

The main advantage of this technique over other existing test methods like bulk statistical nano indentation, is that it really provides a direct correlation between chemical composition structure and local mechanical properties. In regions of interest, though, we've applied this method really to study the spatial distribution and chemical composition structure and mechanical properties in structural biomaterials. It also has many applications including cement-based materials, multi-phase composites, and really any type of heterogeneous material that exhibits that heterogeneity at micrometer and nanometer length scale.

To examine a transverse section of a fish scale short axis, hold the fish scale in a 32 millimeter diameter sample mold, using a commercially available plastic sample holder to keep the sample oriented correctly while mounting in the epoxy. Once the sample is held in the mold, pour an uncured epoxy on the sample. After allowing the epoxy to cure according to the manufacturer's directions, section the mounted sample using a diamond blade high precision cutoff saw at the midline of the sample.

Once the sample has been loaded into the nano and dent enter, use the software controls to move the specimen to the location for the first indent, perform four parallel rows of indents spaced 15 micrometers apart to obtain a statistically significant dataset starting at this location. Then set the nano indenture to a maximum load of five millinewtons loading and unloading rates of 0.1 millinewtons per second, a whole time of 30 seconds, and a minimum indent spacing of five micrometers for each row. When the batch is completed, half the nano indenture create fiducial indents with a maximum load of 100 millinewtons at the first and last indent, which should be in the epoxy before the anoe layer and after the bone layer respectively.

After nano indentation, place the sample back in the PBS solution to avoid further dehydration. Rigidly mount the sample so that the longest dimension is parallel to the detector. After setting up the scanner, secure the mounted sample to the scanner stage using materials that are transparent to x-rays such as styrofoam and paraform.

Position the sample so that it will be in the center of rotation. Throughout the scan, select the highest resolution that allows the entire scale to be in the field of view, which in this case is 7.5 micrometers. Following data acquisition, reconstruct the x-ray projection images to create a data set containing cross-sectional images.

Then use the software to obtain the final 3D gray scale image. Adjust the gray scale range to an appropriate level to remove the artifacts from the styrofoam and paraform For SEM or scanning electron microscopy imaging of nano indents on the polished sample, affix the sample to an SEM stub using double-sided carbon tape with the indented surface facing up. Next, place the specimen into the SEM chamber and pump the chamber into low vacuum mode.

Adjust the working distance to between 3.0 and 5.0 millimeters. Activate the high voltage and navigate to the regions of interest on the specimen, which in this case are the structures present in the anoe and bone layers. Then obtain images at between five kilovolts and 15 kilovolts high voltage and a lower beam current of 3.9 nano amps.

To improve the resolution, capture images from at least three regions of interest at 250 x to 10, 000 x magnifications using the low vacuum back scattered electron BSE detector to aid an identification of changes in bio mineral content and density. Following this, navigate to a region of interest on the polished specimen that includes a nano indentation grid indicated by fiducial marks at the end of each line of indents. Ensure that the high voltage is at least 15 kilovolts.

The beam current is at least 3.9 nano amps, and the working distance is greater than 5.0 millimeters. Then capture the back scattered electron image of the region to be analyzed using EDX. Using EDX analysis software.

Capture the same image to aid in locating areas to perform chemical analysis along the line of indents. Next, using the line analysis technique, position a line to perform chemical analysis along the line of interest of indents starting at the first indent and ending at the last indent. Specify the number of analysis points to be placed along the line using the same number as the number of indents present to provide a direct spatial correlation between the chemical composition and mechanical properties.

When the line is positioned and the points are specified correctly. Initiate the line analysis using the EDX software following completion of the line analysis. Identify elements of interest to be quantified from the point spectra obtained along the specified line on the polished surface of the specimen.

Once elements of interest are identified, perform a background calibration to account for brem straw lung radiation and other effects. Choose the software's deconvolution analysis option to obtain quantitative analysis on each point along the specified line to quantify the chemical composition at each point. Finally, save the quantitative chemical analysis results along with the image of the specified line that was analyzed to aid in spatial correlation with mechanical properties measured using nano indentation in the anoe layer, the nano indenture calculated an average modulus of 69.0 giga pascals and hardness of 3.3 giga Pascals.

The nano indenture determined an average modulus of 14.3 giga pascals and hardness of 0.5 giga pascals for the bone layer. The anoe and bone layer contains quantifiable differences in chemical composition. The carbon spike in the bone layer may be attributed to low mineralization, which results in a carbon increase and a decrease in BSE image brightness, FTIR Spectra of the bone and ENE layers show hydroxyapatite signatures in the outer gwe layer and collagen signatures in the inner bone layer.

X-ray computed tomography shows that the gwe layer does not cover the bone layer where the scales overlap. The brighter gray gwe layers are denser, harder and stiffer while darker gray bone layers are less dense and less stiff. Clear pits near the ging layer center are observed, which demonstrate its non-uniformity.

The lower magnification SEM image of the etched fracture surface revealed nanostructures organized in a layered pattern for the ging layer, which correlates to the FTIR higher magnification SEM, images of the etched fracture surface show oriented nano rods in the ging layer while a fiber like nanostructure is observed in the bone layer After its development. This technique paved the way for additional research on biomaterials and composite materials to study the spatial distribution of chemical composition structure and mechanical properties, and also looking at interfaces in multi-phase composite materials. Alright, after watching this video, you should have a good understanding of how to prepare specimens and perform analyses to spatially correlate chemical composition with structure and mechanical properties in structural biomaterials.

These techniques are also applicable and adaptable for studying other types of heterogeneous multi-phase materials.