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].

<ol>
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The authors have nothing to disclose.

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 constant for steps 2, 4, and 5 of the protocol. For this reason, the two calibration steps (steps 2 and 4 of the protocol) should be performed immediately before the bending test (step 5 of the protocol).
In steps 1.9 and 3.7 of the protocol the stage is leveled with respect to the surface of the isolation table. These steps ensure that <img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg" /> is the component of force perpendicular to the spicule&#39;s longitudinal axis. The frame of the mechanical testing device is manufactured so that the cantilever, LP mirror, and surface of the FODS are all parallel to the surface of the isolation table. This means that the force sensor will measure the component of force and displacement normal to the isolation table surface. If the top of the stage is misaligned by an angle&#160;<img alt="Equation 53" src="/files/ftp_upload/56571/56571eq53.jpg" /> with respect to the surface of the isolation table, then the measured displacement of the LPT will be <img alt="Equation 55" src="/files/ftp_upload/56571/56571eq55.jpg" />, where <img alt="Equation 54" src="/files/ftp_upload/56571/56571eq54.jpg" /> is the actual displacement in the direction perpendicular to the spicule&#39;s longitudinal axis (see Figure 7). Since <img alt="Equation 56" src="/files/ftp_upload/56571/56571eq56.jpg" />, this results in an over prediction of applied forces and the under prediction of spicule displacements per equations (1) and (2).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56571/56571fig7.jpg" /> 
Figure 7: Effect of stage leveling on displacement measurements. (A) The stage is tilted at an angle, <img alt="Equation 53" src="/files/ftp_upload/56571/56571eq53.jpg" />, with respect to the surface of the isolation table and the bottom surface of the cantilever. (B) The displacement of the LP in the vertical direction, <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg" /> (see Figure 1 (D)), is measured by the FODS. The component of the LP displacement in the direction perpendicular to the spicule&#39;s axis is <img alt="Equation 54" src="/files/ftp_upload/56571/56571eq54.jpg" />. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56571/56571fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In steps 5.1-5.3 of the protocol the LPT is positioned mid way across the trench&#39;s span. Misalignment of the LPT with respect to the mid span will result in the specimen appearing stiffer than it actually is 31,32. That is, the spicule&#39;s displacement will be smaller than that which would be measured if the same force were applied at the mid span. This type of misalignment can be avoided by not removing the stage from the stage base plate or adjusting the x-axis stage position after the centering procedure is complete (steps 5.1-5.3 of the protocol).
One limitation of this method is that in order to reduce the relative measurement uncertainty of the force and displacement measurements, the cantilever stiffness should be selected so that the FODS output voltages span the full range of 1.8 to 4.5 V during the bending test. However, this voltage range corresponds to a cantilever displacement of approximately &#8776;250 &#181;m, which is roughly the same as the spicule displacement just before it fails (see Figure 6 (C)). This means that the cantilever and the spicule have similar stiffnesses. While this is not problematic for measuring the elastic response and strength properties of the spicules, it does preclude the accurate measurement of the spicules&#39; toughness properties. This is because in order to ensure accurate measurement of toughness properties, a crack in the spicule must propagate in a controlled manner 33. Typically, this is only possible if the testing device is much stiffer than the specimen 33. In order to increase the stiffness of the testing device, a more sensitive displacement sensor could be used in place of the FODS.
While the bending test protocol is demonstrated on E. aspergillum spicules, the mechanical testing device can be used to perform three-point bending tests on other LBBSs and synthetic materials as well. This mechanical testing device is most appropriate for specimens whose cross-sectional diameters range from 0.01 to 1 mm and for trench spans ranging from 1 to 10 mm. For larger diameters, the sample stage should be redesigned so that the specimen cannot roll across the stage. This is not an issue for smaller fibers, like the spicules, because the roughness of the stage&#39;s surface is enough to prevent the specimen from rolling. The radii of the trench edges and LPT should also be made larger to avoid introducing local damage at the points where the specimen is supported 31,32. Furthermore, the stage leveling plate should be fastened to the stage base plate (see Figure 4A) using &#188;&#34;-20 socket head cap screws after step 3.7 of the protocol to prevent stage tilting if forces exceed &#8776;1 N.
For accurate force and displacement measurement, the cantilever&#39;s stiffness should always be much smaller than the frame&#39;s stiffness (&#8776;107 N/m). This requirement limits the maximum force that can be applied by this device to &#8776;25 N. Consequently, it is important to estimate the maximum force a specimen can withstand before performing a bending test to determine if this device can be used to perform the test.
This work provides the protocol, technical drawings (see Supplementary File 1) and software (see Supplementary Code files) for reproducing and using our mechanical testing device. This will hopefully provide a platform for accurately measuring the flexural behavior of many different LBBSs. These measurements are a prerequisite for developing a deeper understanding the relationship between a LBBS&#39;s architecture and its mechanical properties.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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" /> direction, <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" />. However, for the mechanical testing device described here, several post-processing steps must be performed to transform the output data obtained from steps 2, 4 and 5 of the protocol into this desired <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />- <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> data. The data files obtained from the three-point bending test are: 1) the voltage-displacement interpolation file; 2) the force calibration file; and 3) the bending test file. A summary of the measured and derived quantities is shown in Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Symbol</td>
			<td colspan="4">Definition</td>
		</tr>
		<tr>
			<td>Nh</td>
			<td colspan="4">Number of voltages values in the voltage-displacement interpolation output file</td>
		</tr>
		<tr>
			<td>Vh</td>
			<td colspan="4">Measured voltage values in step 4 of the Protocol</td>
		</tr>
		<tr>
			<td>&#963;Vh</td>
			<td colspan="4">Standard deviation of Vh</td>
		</tr>
		<tr>
			<td>zsh</td>
			<td colspan="4">Measured stage position in step 4 of the Protocol</td>
		</tr>
		<tr>
			<td>Nc</td>
			<td colspan="4">Number of force measurements in the force calibration output file</td>
		</tr>
		<tr>
			<td>Fc</td>
			<td colspan="4">Force applied by calibration weights in step 2 of the Protocol</td>
		</tr>
		<tr>
			<td>Vc</td>
			<td colspan="4">Measured voltage values in step 2 of the Protocol</td>
		</tr>
		<tr>
			<td>&#963;Vc</td>
			<td colspan="4">Standard deviation of Vc</td>
		</tr>
		<tr>
			<td>zlc</td>
			<td colspan="4">Position of the LP in step 2 of the Protocol computed using Vh and Vc</td>
		</tr>
		<tr>
			<td>wlc</td>
			<td colspan="4">Displacement of the LP in step 2 of the Protocol computed from zlc</td>
		</tr>
		<tr>
			<td>Nt</td>
			<td colspan="4">Number of force and displacement measurements in the bending test output file</td>
		</tr>
		<tr>
			<td>zst</td>
			<td colspan="4">Position of the stage in step 5 of the Protocol</td>
		</tr>
		<tr>
			<td>wst</td>
			<td colspan="4">Displacement of the stage in step 5 of the Protocol</td>
		</tr>
		<tr>
			<td>Vt</td>
			<td colspan="4">Measured voltage values in step 5 of the Protocol</td>
		</tr>
		<tr>
			<td>&#963;Vt</td>
			<td colspan="4">Standard deviation of Vt</td>
		</tr>
		<tr>
			<td>zlt</td>
			<td colspan="4">Position of the LP in step 5 of the Protocol computed using Vh and Vt</td>
		</tr>
		<tr>
			<td>wlt</td>
			<td colspan="4">Displacement of the LP in step 5 of the Protocol computed from zlt</td>
		</tr>
		<tr>
			<td>F</td>
			<td colspan="4">Force applied by the LP in step 5 of the Protocol computed from zlt</td>
		</tr>
		<tr>
			<td>w0</td>
			<td colspan="4">Displacement of the spicule&#8217;s cross-section under the LP in step 5 of the Protocol</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of symbols used for quantities measured in steps 2, 4 and 5 of the Protocol and computed in the Representative Results section.
The purpose of the voltage-displacement interpolation file is to relate measured FODS output voltages to LPT displacements. This is done by rigidly coupling the LPT to the translation stage so that as the stage is moved in the <img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg" /> direction, the change in the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" />-axis stage position is equal to the LPT displacement (step 4 of the protocol). The voltage-displacement interpolation file contains a set of points <img alt="Equation 15" src="/files/ftp_upload/56571/56571eq15.jpg" />, where&#160;<img alt="Equation 16" src="/files/ftp_upload/56571/56571eq16.jpg" /> is the average FODS output voltage taken over 100 measurements at a sampling rate of 1000 Hz, <img alt="Equation 17" src="/files/ftp_upload/56571/56571eq17.jpg" /> is the associated standard deviation of the 100 voltage measurements, <img alt="Equation 18" src="/files/ftp_upload/56571/56571eq18.jpg" /> is the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" />-axis stage position and&#160;<img alt="Equation 19" src="/files/ftp_upload/56571/56571eq19.jpg" /> is the number of stage displacement steps (see Figure 6 (B)).
The force calibration file allows the cantilever stiffness to be measured so that LP displacements can be used to compute the magnitude of the force applied by the LP. The force calibration file contains a set of points <img alt="Equation 20" src="/files/ftp_upload/56571/56571eq20.jpg" />, where&#160;<img alt="Equation 21" src="/files/ftp_upload/56571/56571eq21.jpg" /> is the average FODS output voltage taken over 100 measurements at a sampling rate of 1000 Hz,&#160;<img alt="Equation 22" src="/files/ftp_upload/56571/56571eq22.jpg" /> is the associated standard deviation of the 100 voltage measurements,&#160;<img alt="Equation 23" src="/files/ftp_upload/56571/56571eq23.jpg" /> is the force exerted by the weights on the LPT, and&#160;<img alt="Equation 24" src="/files/ftp_upload/56571/56571eq24.jpg" /> is the number of calibration weights used. Notice that there are two more points than there are calibration weights because the first point is measured for zero applied force and the second point for the force exerted by the wire hook alone.
Finally, the bending test file is used to compute&#160;<img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> and <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />. It contains a set of points <img alt="Equation 25" src="/files/ftp_upload/56571/56571eq25.jpg" />, where <img alt="Equation 26" src="/files/ftp_upload/56571/56571eq26.jpg" /> is the average FODS output voltage taken over 100 measurements at a sampling rate of 1000 Hz, <img alt="Equation 27" src="/files/ftp_upload/56571/56571eq27.jpg" /> is the associated standard deviation of the 100 voltage measurements, <img alt="Equation 28" src="/files/ftp_upload/56571/56571eq28.jpg" /> is the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" />-axis stage position and&#160;<img alt="Equation 29" src="/files/ftp_upload/56571/56571eq29.jpg" /> is the number of stage displacement steps during the bending test.
First, the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT&#39;s position during the force calibration, <img alt="Equation 30" src="/files/ftp_upload/56571/56571eq30.jpg" />, is found by using the set&#160;<img alt="Equation 31" src="/files/ftp_upload/56571/56571eq31.jpg" /> to map <img alt="Equation 21" src="/files/ftp_upload/56571/56571eq21.jpg" />values to <img alt="Equation 32" src="/files/ftp_upload/56571/56571eq32.jpg" /> values via linear interpolation. The <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT displacement is given by <img alt="Equation 33" src="/files/ftp_upload/56571/56571eq33.jpg" />, <img alt="Equation 34" src="/files/ftp_upload/56571/56571eq34.jpg" />. Since the LPT displacements are small compared to the length of the cantilever, the relationship between <img alt="Equation 23" src="/files/ftp_upload/56571/56571eq23.jpg" /> and <img alt="Equation 35" src="/files/ftp_upload/56571/56571eq35.jpg" /> appears to be linear. Therefore, the cantilever stiffness can be computed by fitting a line to the <img alt="Equation 36" src="/files/ftp_upload/56571/56571eq36.jpg" /> data and computing the slope, <img alt="Equation 37" src="/files/ftp_upload/56571/56571eq37.jpg" />. A representative set of points <img alt="Equation 36" src="/files/ftp_upload/56571/56571eq36.jpg" />and its corresponding fitted line are shown in Figure 6A. The stiffness of the cantilever used in the bending experiments was 90.6 &#177; 0.3 N/m.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56571/56571fig6.jpg" /> 
Figure 6: Representative results of the three-point bending test. (A) Force and displacement data (gray) obtained in step 2 of the protocol along with the linear fit (blue) used for estimating the stiffness of the cantilever. (B) Representative example of the data contained within the voltage-displacement interpolation output file. For a measured FODS output voltage, <img alt="Equation 51" src="/files/ftp_upload/56571/56571eq51.jpg" />, the position of the stage, <img alt="Equation 52" src="/files/ftp_upload/56571/56571eq52.jpg" />, can be obtained via linear interpolation. This is used to measure the cantilever displacement, <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg" />, during the bending test. (C) Representative force-displacement responses of 3 different E. aspergillum anchor spicules from successful three-point bending tests. (D) A force-displacement response from an unsuccessful three-point bending test. The nonlinearity of the curve suggests that the spicule was not properly seated on the sample stage and slid or reoriented after initial contact was made with the LPT. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56571/56571fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT&#39;s position during the bending test, <img alt="Equation 38" src="/files/ftp_upload/56571/56571eq38.jpg" />, is found by using the set <img alt="Equation 31" src="/files/ftp_upload/56571/56571eq31.jpg" /> to map <img alt="Equation 26" src="/files/ftp_upload/56571/56571eq26.jpg" /> values to <img alt="Equation 39" src="/files/ftp_upload/56571/56571eq39.jpg" /> values via linear interpolation. The <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT displacement during the bending test is given by <img alt="Equation 40" src="/files/ftp_upload/56571/56571eq40.jpg" />,&#160;<img alt="Equation 41" src="/files/ftp_upload/56571/56571eq41.jpg" />. The <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the stage displacement during the bending test is given by <img alt="Equation 42" src="/files/ftp_upload/56571/56571eq42.jpg" />.
Since the LPT and the spicule are in contact during the entirety of the bending test, the spicule displacement,&#160;<img alt="Equation 43" src="/files/ftp_upload/56571/56571eq43.jpg" /> is given by
<img alt="Equation 44" src="/files/ftp_upload/56571/56571eq44.jpg" /> (1)
and the force applied by the LPT, <img alt="Equation 45" src="/files/ftp_upload/56571/56571eq45.jpg" />, is
<img alt="Equation 46" src="/files/ftp_upload/56571/56571eq46.jpg" /> (2)
It is important to note that since the set <img alt="Equation 31" src="/files/ftp_upload/56571/56571eq31.jpg" /> is used to obtain both <img alt="Equation 32" src="/files/ftp_upload/56571/56571eq32.jpg" /> and <img alt="Equation 39" src="/files/ftp_upload/56571/56571eq39.jpg" /> values via interpolation, the values of the <img alt="Equation 47" src="/files/ftp_upload/56571/56571eq47.jpg" /> and <img alt="Equation 26" src="/files/ftp_upload/56571/56571eq26.jpg" /> must be within the range of <img alt="Equation 16" src="/files/ftp_upload/56571/56571eq16.jpg" />. This is ensured by setting appropriate values for the starting voltage and high voltage stop values in steps 2, 4 and 5 of the protocol.
Figure 6C shows force-displacement curves for three representative spicules. For slender, linear elastic structures loaded in three-point bending, <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" /> is expected to increase linearly with <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> for small values of <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> 30. Nonlinearity of the <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />- <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> curve for small <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> (e.g., see Figure 6D) typically suggests that the spicule may not be seated correctly on the sample stage. In this case, the test should be stopped and the spicule repositioned on the sample stage (step 3.6 of the protocol).
In order to ensure sufficient accuracy of the <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" /> and <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> measurements, the total voltage change over the course of the bending test, <img alt="Equation 48" src="/files/ftp_upload/56571/56571eq48.jpg" />, should be at least 1 V. If the total voltage change is less than 1 V, a more compliant cantilever should be selected.

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 ...

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8.4K Views.  Brown University. 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 µN up to 10 N and can therefore accommodate a variety of fiber sizes.

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

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...