A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

The overall goal of this experiment is to measure the flexural behavior of fibers whose diameters are between 10 and 100 micrometers. This method can help answer key questions about the mechanical behavior of biological structures, such as the strength and stiffness properties of marine sponge spicules. The main advantage of this technique is that it can be used to measure the mechanical behavior of a wide variety of materials with different sizes and elastic properties.

Though this method can provide insight into the mechanical behavior of spicules, it can also be applied to other loading-bearing biological structures, such as plant stems and feather rachises. To begin, attach the load point to the cantilever, using number 4-40 socket head cap screws. Take care not to plastically deform the cantilever arms while attaching the load point.

Then, position the load point tip away from the cantilever plate, and loosely attached the cantilever to the plate, using number 6-32 socket head cap screws. Next, insert the 1/8-inch alignment pins through the cantilever and plate, tighten the screws, and then remove the alignment pins. Retract the fiber optic displacement sensor as much as possible by turning the sensor micrometer counterclockwise.

Then, loosely attach the cantilever plate to the frame, using number 6-32 socket head cap screws with the load point tip pointing in the negative z-direction. Again, insert the 1/8-inch alignment pins, this time through the frame and cantilever plate, tighten the screws, and then remove the alignment pins. Now, seat the stage on the stage baseplate so that the tips of the micrometer heads on the leveling plate rest in the stage base plate divots.

Place a bubble level on the isolation table, and adjust the pressure in each of the table’s legs by turning the valve arm thumb screws so that the surface is level. Move the bubble level to the top of the stage leveling plate, and adjust the micrometer so that it is also level. Note the micrometer positions, and remove the stage from the stage baseplate.

Use a pair of tweezers to grasp one anchor spicule by its distal end, and pull to remove it from the skeleton. Place the spicule on a clean microscope slide. Using a quintuple zero size red sable brush, hold the spicule against the slide.

Cut a four millimeter section of the spicule by pushing a razor blade against the spicule on either side of the brush, perpendicular to the slide surface. Then, discard the distal and proximal spicule sections, and keep the four millimeter section cut from the midpoint. Transfer the spicule section to the sample stage.

Position it across the trench with the desired span for the bending test, and gently push it in the positive y-direction against the trench ridge to ensure that the spicule is perpendicular to the trench edges. Seat the stage on the stage baseplate so that the tips of the micrometer spindles rest in the stage baseplate divots. If needed, adjust the micrometers on the stage leveling plate.

Open the Bending Test program found in the supplementary code file, and set the Step Size to two micrometers, the Maximum Displacement to 0.5 millimeters, the Low Voltage Stop to 1.5 volts, and the High Voltage Stop to 4.6 volts, using the text boxes shown in the user interface. Select the desired image, data directories, and the output file name, using the text boxes in the user interface. Then, set the Save Images switch in the user interface to the down position, and click the green rectangular button below the words Voltage Difference so that it becomes illuminated.

Now, run the Bending Test program, and wait for the motor controller and camera interfaces to initialize. Turn on the illuminator, and adjust the brightness so that the load point tip is visible. Then, turn the fiber optic displacement sensor micrometer clockwise until the output voltage displayed in the user interface graph is about 1.7 volts.

Now, use the potentiometer slider on the z-axis motor controller to move the stage in the positive z-direction until it is about one centimeter below the load point tip, and set the z-axis home position by clicking the Home button. Use the potentiometer sliders on the x-and y-axis motor controllers to position the load point tip over the center of the thin steel strip located on the sample stage in the negative x-direction from the trench. Then, use the potentiometer slider on the z-axis motor controller to move the stage in the positive z-direction until the stage is within the microscope’s field of view.

Click the button labeled Begin Test, and when prompted, enter values of 0.003 volts and 0.001 millimeters for touch sensitivity and touch-off step size, respectively. Click OK, and wait several minutes for the calibration step to finish. Open and run the Basic Data program found in the supplementary code file, and turn the fiber optic displacement sensor micrometer counterclockwise until the output voltage displayed on the user interface graph is approximately three volts.

Then, use the potentiometer slider on the x-axis motor controller to position the load point tip between the trench edges above the spicule. Also, use the potentiometer slider on the z-axis motor controller to move the stage in the positive z-direction until the load point tip is below the top surface of the trench ridge. Finally, use the potentiometer slider on the y-axis motor controller to bring the front surface of the trench ridge into focus so that the complete width of the load point tip is between the edges of the trench ridge.

Then, stop the Basic Data program by clicking the Stop button. Next, open and run the Center Load Point program, as found in the supplementary code file. Use the x-axis motor controller to move the stage until the load point tip is nearly in contact with the right trench edge.

Then, click on the Find Edge button. When prompted, use the x-axis motor controller to move the stage until the load point tip is nearly in contact with the left trench edge. At this point, click on the Find Edge button again, and wait for the program to position the load point tip midway across the trench span.

Next, open the Bending Test program. Set the Step Size to two micrometers, Maximum Displacement to 0.5 millimeters, Low Voltage Stop to 1.5 volts, and High Voltage Stop to 4.5 volts using the text boxes in the user interface. Additionally, select the desired image and data directories and the output file name, using the text boxes in the user interface.

Set the Save Images switch in the user interface to the up position, and click the green rectangular button below words Voltage Difference so that it is not illuminated. Then, run the Bending Test program, and wait for the motor controller and camera interfaces to initialize. Once initialized, move the stage in the positive z-direction, using the potentiometer slider on the motor controller, until the spicule is within the microscope’s field of view.

Then, user the potentiometer slider on the y-axis motor controller to move the stage until the spicule is under the load point tip. Next, adjust the microscope focus knob so that the spicule is in focus in the user interface. Then, turn the fiber optic displacement sensor micrometer counterclockwise until the output voltage is approximately 1.8 volts.

Once set, click on Begin Test, and wait until the bending test is completed and the stage returns to the z-axis home position. The displacement of the spicule in the z-direction and the force applied by the load point tip can be computed using the voltage-displacement interpolation file, the force calibration file, and the bending test file obtained from the three-point bending test. The voltage-displacement interpolation file is used to measure the cantilever displacement during the bending test.

To estimate the stiffness of the cantilever, the force calibration is used, which is then used to relate the cantilever displacement to the force applied by the load point tip. Taken together, these can be used to create the force displacement responses. Shown here, are three different E.aspergillum anchor spicules from successful three-point bending tests.

Once mastered, a bending test can be performed with this device in approximately 10 to 15 minutes. The most important aspect of this procedure is ensuring that the spicule is properly seated on the stage and that its axis is perpendicular to the trench edges. Three-point bending tests provide a relatively simple way for researchers studying load-bearing biological structures to gain insight into their mechanical behavior.

Following the bending test, beam theories can be used to compute the spicules Young’s modulus and fracture strength from the force displacement data.