Name: a millimeter scale flexural testing system for measuring the mechanical properties of marine sponge spicules
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Description: Watch this Scientific Journal Video about A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules at JoVE.com

This work was supported by National Science Foundation [Mechanics of Materials and Structures Program, grant number 1562656]; and the American Society of Mechanical Engineers [Haythornthwaite Young Investigator Award].

1. Assembly and Alignment
<ol>
	<li>Choose a cantilever whose stiffness is appropriate for the intended experiment. Attach the LP to the cantilever using #4-40 socket head cap screws (SHCSs) (see Figure 3A). Take care to not plastically deform the cantilever arms while attaching the LP.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56571/56571fig3.jpg" /> 
Figure 3: Procedure for ...

<ol>
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Several steps of the protocol are particularly important for ensuring that forces and displacements are measured accurately. While some of these critical steps are universal to all three-point bending tests, others are unique to this mechanical testing device.
In step 1.2 of the protocol the LP mirror is cleaned and inspected for scratches, and in step 1.6 of the protocol the FODS gain is set. It is important for the gain and the LP mirror reflectance to be co...

By studying the architectures of load bearing biological structures (LBBSs), such as shell and bone, engineers have developed new composite materials that are both strong and tough 1. It has been shown that the remarkable mechanical properties of LBBSs and their bio-inspired counterparts are related to their intricate internal architectures 2. However, the relationships between LBBS architectures and mechanical properties are not fully understood. Measuring a LBBS&#39;s mechanical response is the first step toward understanding how its architecture enhances its mechanical properties.
However, it is important that the type of test used to measure a LBBS&#39;s mechanical response is consistent with its mechanical function. For example, since feathers must support aerodynamic loads, the primary function of a feather rachis is to provide flexural stiffness 3. Therefore, a bending test is preferred to a uniaxial tension test for measuring its mechanical response. In fact, many LBBSs&#8212;such as feather rachises 3, grass stems 4, and spicules 5,6,7,8&#8212;primarily deform by bending. This is because these LBBSs are slender&#8212;i.e., their length is much greater than their width or depth. However, performing bending tests on these LBBSs is challenging because the forces and displacements that they can withstand before failing range from 10-2 to 102 N and 10-4 to 10-3 m, respectively 3,4,5,7,8. Consequently, the device used to perform these mechanical tests should have force and displacement resolutions of &#8776;10-5 N and &#8776;10-7 m (i.e., 0.1% of the sensor&#39;s maximum measureable force and displacement), respectively.
Commercially available, large scale, mechanical testing systems typically cannot measure forces and displacements with this resolution. While atomic force microscope-based 9,10 or microelectromechanical systems-based 11 testing devices have adequate resolution, the maximum force (respective displacement) they can measure is smaller than the maximum force (respective displacement) that the LBBS can withstand. Therefore, to perform bending tests on these LBBSs, engineers and scientists must rely on custom-built mechanical testing devices 5,7,12,13. The primary advantage of these custom-built devices is that they can accommodate large ranges of forces and displacements. However, the construction and operation of these devices is not well documented in the literature.
A protocol is described for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. Technical drawings, including all dimensions, of the components of the mechanical testing device are provided in the Supplementary Material. The primary advantage of this mechanical testing device is that the force and displacement ranges can be easily adjusted to suit different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope 9. In this device, a specimen is placed across a trench cut in a stainless steel plate (see Figure 1A-C). The span of the trench is measured from optical micrographs to be 1278 &#177; 3 &#181;m (mean &#177; standard deviation; n = 10). The trench edges support the specimen during a bending test (see Figure 1C, and D). This sample stage is attached to a three-axis translation stage and positioned beneath an aluminum wedge so that the wedge is located midway across the trench&#39;s span (see Figure 1C). By moving the stage in the <img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg" /> direction (see Figure 1A, and C), the specimen is pushed into the wedge causing the specimen to bend.
We refer to the wedge as the load point tip (LPT) and the component of the device that contains the wedge as the load point (LP). The LP is attached to the end of a cantilever whose displacement is measured by a fiber optic displacement sensor (FODS). The FODS emits infrared light, which is reflected off of a mirror located on the top surface of the LP (see Figure 1B) and received by an optical fiber in the FODS. A &#8776;5 mm square piece of a polished silicon wafer is used as the LP mirror and is affixed to the LP using epoxy. The FODS measures displacements by comparing the intensities of the emitted and reflected light. The cantilever stiffness and displacement are used to compute the force,&#160;<img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg" />, experienced by the wedge due to its interaction with the specimen. The cantilever displacement is also used to compute the displacement of the specimen&#39;s cross-section beneath the wedge, <img alt="Equation 3" src="/files/ftp_upload/56571/56571eq3.jpg" />. Cantilever-based force sensors have been used in a number of micro- and macro-scale mechanical testing studies 10,11,12,13,14. The specific design presented here is adapted from a mechanical testing device used for performing adhesive contact experiments 14. A similar design has also been used in a commercially available micro-tribometer 15,16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56571/56571fig1.jpg" /> 
Figure 1: Overview of the custom-built mechanical testing device. (A) A computer aided design rendering of the device. The stage components are highlighted in green. The force sensing subassembly (cantilever, load point (LP)) is highlighted in red. (B) A magnified view of (A). The LP mirror is shown in blue on the top surface of the LP beneath the FODS and is labeled LPM. (C) The coordinate system used to describe the motion of the translation stage. By leveling the stage in step 1.9 of the protocol, the&#160;<img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg" /> direction is made to coincide with the vector normal to the surface of the LP mirror. (D) A schematic of the three-point bending configuration showing the deformation of the spicule and the measured displacements <img alt="Equation 49" src="/files/ftp_upload/56571/56571eq49.jpg" />, and <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg" />. <a href="//cloudflare.jove.com/files/ftp_upload/56571/56571fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum6,7. This sponge&#39;s skeleton is an assembly of filaments, called spicules (see Figure 2A). The spicules are &#8776;50 &#181;m thick and are composed primarily of silica 6. Biosilica-based spicules are found in sponges belonging to the classes Demospongiae, Homoscleromorpha, and Hexactinellida. Sponges, such as E. aspergillum, that belong to the class Hexactinellida are also known as &#34;glass sponges.&#34; While the spicules of glass sponges are composed primarily of silica, it has been shown that the silica often contains an organic matrix composed of either collagen 17,18 or chitin 19,20,21. This organic matrix plays an important role in silica biomineralization 18,20. Furthermore, in some spicules the organic matrix also serves as a template for the biomineralization of calcium 22. In addition to being distributed within the silica, the organic matrix can also form distinct layers that partition the spicule&#39;s silica into concentric, cylindrical lamellae 6,23. It has been shown that this concentric, lamellar architecture can affect the spicules&#39; deformation behavior 6,7,8,24,25,26. Consequently, the spicules&#39; mechanical properties are determined by a combination of their chemistry (i.e., the chemical structure of the silica-protein composite) and their architecture 27. Both the chemical structure and architecture of glass sponge spicules are still under investigation 24,28,29.
Most of the spicules in E. aspergillum are cemented together to form a stiff skeletal cage. However, at the base of the skeleton there is a tuft of very long (&#8776;10 cm) spicules known as the anchor spicules (see Figure 2A). We describe the protocol for performing three-point bending tests on small sections of the anchor spicules.
In step 1 of the protocol, the procedure for assembling and aligning the components of the custom-built mechanical testing device is described. Steps 2 and 4 of the protocol provide instructions for generating calibration data used to compute forces and displacements in the bending test. The steps taken to prepare a section of a spicule and mount it to the test fixture are described in step 3. The procedure for conducting the bending test on the spicule section is described in step 5. Finally, in the Representative Results section the calibration data obtained in steps 2 and 4 are used along with the bending test data obtained in step 5 to compute <img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/56571/56571eq3.jpg" />.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56571/56571fig2.jpg" /> 
Figure 2: Procedure for sectioning and inspecting E. aspergillum spicules. (A) The skeleton of E. aspergillum. The tuft of free-standing anchor spicules is shown at the base of the skeleton. The scale bar is ~25 mm. (B) A single anchor spicule is held in place on a microscope slide using a #00000 red sable brush and sectioned using a razor blade. The scale bar is ~12 mm. (C) A section of an E. aspergillum spicule placed across the trench on the sample stage. The trench edges and trench ridge are highlighted in teal and orange, respectively. The spicule is pushed against the trench ridge to ensure that its axis is perpendicular to the trench edges. (D) A micrograph of a spicule that passes the inspection procedure described in step 3.4 of the protocol, which describes how to determine if a spicule section is damaged and should be discarded. (E) A micrograph of a spicule containing many cracks and missing large sections of silica layers that would fail the inspection procedure described in step 3.4 of the protocol. Scale bars =&#160; 250 &#181;m (C), 100 &#181;m (D), and 100 &#181;m&#160;(E). <a href="//cloudflare.jove.com/files/ftp_upload/56571/56571fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>TMC 36&#34; x 48&#34; isolation table with 4&#34; CleanTop breadboard</td>
			<td>TMC</td>
			<td>63-563</td>
			<td>Isolation Table</td>
		</tr>
		<tr>
			<td>Diffeential Screw Adjuster</td>
			<td>Thorlabs</td>
			<td>DAS110</td>
			<td>For stage leveling plate</td>
		</tr>
		<tr>
			<td>1&#34; Travel Micrometer Head with 0.001&#34; Graduations</td>
			<td>Thorlabs</td>
			<td>150-801ME</td>
			<td>For stage leveling plate</td>
		</tr>
		<tr>
			<td>Right-Angle Bracket for PT Series Translation Stages, 1/4&#34;-20 Mounting Holes</td>
			<td>Thorlabs</td>
			<td>PT102</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>1&#34; Dovetail Translation Stage, 1/4&#34;-20 Taps</td>
			<td>Thorlabs</td>
			<td>DT25</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>1&#34; Translation Stage with 1/4&#34;-170 Adjustment Screw, 1/4&#34;-20 Taps</td>
			<td>Thorlabs</td>
			<td>PT1B</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>12&#34; Length, Dovetail Optical Rail</td>
			<td>Edmund Optics</td>
			<td>54-401</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>2.5&#34; Width, Dovetail Carrier</td>
			<td>Edmund Optics</td>
			<td>54-404</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>0.5&#34; Width, Dovetail Carrier</td>
			<td>Edmund Optics</td>
			<td>54-403</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>InfiniTube Mounting C-Clamp with &#188;-20</td>
			<td>Edmund Optics</td>
			<td>57-788</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Standard (with no In-Line Attachment), InfiniTube</td>
			<td>Edmund Optics</td>
			<td>56-125</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Standard In-Line Attachment (Optimized at 2X-10X), InfiniTube</td>
			<td>Edmund Optics</td>
			<td>56-126</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Mitutoyo/Achrovid Objective Adapter (M26 to M27)</td>
			<td>Edmund Optics</td>
			<td>53-787</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>5X Infinity Achrovid Microscope Objective</td>
			<td>Edmund Optics</td>
			<td>55-790</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>0.316&#34; ID, Fiber Optic Adapter SX-6</td>
			<td>Edmund Optics</td>
			<td>38-944</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>&#188;&#34; x 36&#34;, Flexible Fiber Optic Light Guide</td>
			<td>Edmund Optics</td>
			<td>42-347</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>115V, MI-150 Fiber Optic Illuminator w/IR Filter and Holder</td>
			<td>Edmund Optics</td>
			<td>55-718</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Allied Vision Manta G-223 2/3&#34; Color CMOS Camera</td>
			<td>Edmund Optics</td>
			<td>88-452</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Power Supply for Manta/ Guppy Pro/ Stingray/ Pike</td>
			<td>Edmund Optics</td>
			<td>68-586</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>1/4&#34; Travel Single Axis Translation Stage</td>
			<td>Thorlabs</td>
			<td>MS1S</td>
			<td>FODS micrometer</td>
		</tr>
		<tr>
			<td>Analog Reflectance Dependent Fiber Optic Displacement Sensor</td>
			<td>Philtec</td>
			<td>D20</td>
			<td>FODS</td>
		</tr>
		<tr>
			<td>30V, 3A DC Power Supply</td>
			<td>Agilent</td>
			<td>U8001A</td>
			<td>Power supply for DAQ and FODS</td>
		</tr>
		<tr>
			<td>14-Bit, 48 kS/s Low-Cost Multifunction DAQ</td>
			<td>National Instruments</td>
			<td>USB-6009</td>
			<td>DAQ for FODS</td>
		</tr>
		<tr>
			<td>Three Axis Motorized Translation Stage</td>
			<td>Thorlabs</td>
			<td>Thorlabs T25 XYZ-E/M</td>
			<td>Translation stage</td>
		</tr>
		<tr>
			<td>T-Cube DC Servo Motor Controller</td>
			<td>Thorlabs</td>
			<td>TDC001</td>
			<td>Motor controller for stage</td>
		</tr>
		<tr>
			<td>T-Cube Power Supply</td>
			<td>Thorlabs</td>
			<td>TPS001</td>
			<td>Power supply for motor controller</td>
		</tr>
		<tr>
			<td>National Instruments LabVIEW (2013 SP1)</td>
			<td>National Instruments</td>
			<td></td>
			<td>Used for running software</td>
		</tr>
		<tr>
			<td>National Instruments LabVIEW Vision Acquisition Software (2016)</td>
			<td>National Instruments</td>
			<td></td>
			<td>Used for running software</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ci-POL Main Body</td>
			<td>MVI</td>
			<td>MDA96000</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon Pi Intermediate Tube with Analyzer Slider</td>
			<td>MVI</td>
			<td>MDB45305</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon Dia-Polarizer</td>
			<td>MVI</td>
			<td>MDN11920</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Power Cord - 7&#39;6&#34;</td>
			<td>MVI</td>
			<td>79035</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon P-Amh Mechanical Stage</td>
			<td>MVI</td>
			<td>MDC45000</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon Lwd Achromat Condenser</td>
			<td>MVI</td>
			<td>MBL16100</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon LV-NBD5BD-CH Manual Quint Nosepiece ESD</td>
			<td>MVI</td>
			<td>MBP60125</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon C-TF Trinocular Tube F</td>
			<td>MVI</td>
			<td>MBB93100</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon CFI 10X Eyepiece FN 22mm NC</td>
			<td>MVI</td>
			<td>MAK10110</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon TU Plan Flour BD 10x Objective</td>
			<td>MVI</td>
			<td>MUE42100</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Venus Flower Basket Sponge</td>
			<td>Denis Brand</td>
			<td>N/A</td>
			<td>Sponge skeleton</td>
		</tr>
		<tr>
			<td>3.5X Headband Flip-Up Magnifier</td>
			<td>McMaster Carr</td>
			<td>1490T5</td>
			<td>Used for spicule sectioning</td>
		</tr>
		<tr>
			<td>&#216;1&#34; Silicon Wafer, Type P / &#60;100&#62;</td>
			<td>Ted Pella</td>
			<td>16011</td>
			<td>Used for load point mirror</td>
		</tr>
		<tr>
			<td>Low Lint Tapered Tip Cotton Swab</td>
			<td>McMaster Carr</td>
			<td>71035T31</td>
			<td>Used for cleaning LP mirror</td>
		</tr>
		<tr>
			<td>Rubber grip precision knife</td>
			<td>McMaster Carr</td>
			<td>35575A68</td>
			<td>Used for sectioning spicules</td>
		</tr>
		<tr>
			<td>Microscope Slides, frosted end, 75 x 25 x 1mm</td>
			<td>Ted Pella</td>
			<td>260409</td>
			<td>Used for sectioning spicules</td>
		</tr>
		<tr>
			<td>Sable Brushes, #00000, 0.08mm W x 4.0mm L</td>
			<td>Ted Pella</td>
			<td>11806</td>
			<td>Used for handling spicules</td>
		</tr>
		<tr>
			<td>PELCO Pro High Precision Tweezers, extra fine tips, superior finish</td>
			<td>Ted Pella</td>
			<td>5367-5NM</td>
			<td>Used for handling spicules</td>
		</tr>
		<tr>
			<td>Dual Axis Linear Scale Micrometer</td>
			<td>Edmund Optics</td>
			<td>58-608</td>
			<td>Used for calibrating the microscopes</td>
		</tr>
		<tr>
			<td>FLEX-A-TOP FT-38 CAS</td>
			<td>ESD Plastic Containers</td>
			<td>FT-38-CAS</td>
			<td>Used for storing spicules</td>
		</tr>
		<tr>
			<td>Plastic Vial Bullseye Level</td>
			<td>McMaster Carr</td>
			<td>2147A11</td>
			<td>Used for leveling the stage</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>MS105DU</td>
			<td>Used to mass calibration weights</td>
		</tr>
	</tbody>
</table>

a millimeter scale flexural testing system for measuring the mechanical properties of marine sponge spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

The most basic outputs of any mechanical test are the magnitude of the force applied to the specimen and the displacement at the location where the force is applied. In the case of a three-point bending test, the goal is to obtain the magnitude of the force applied by the LPT, <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />, and the displacement of the specimen&#39;s cross-section beneath the LPT in the <img alt="Equation 4" src="/files/ftp_upload/56571/56571eq4.jpg"...

Watch this Scientific Journal Video about A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules at JoVE.com

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

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

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.

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