Name: flapping soft fin deformation modeling using planar laser-induced fluorescence imaging
Uploaded: 2022-04-28T13:30:00
Description: Watch this Scientific Journal Video about Flapping Soft Fin Deformation Modeling using Planar Laser-Induced Fluorescence Imaging at JoVE.com

This research was supported by the Office of Naval Research through a US Naval Research Laboratory (NRL) 6.2 base program and performed while Kaushik Sampath was an employee of the Acoustics Division at NRL and Nicole Xu held an NRC Research Associateship award in the Laboratories for Computational Physics and Fluid Dynamics at NRL. The authors would like to acknowledge Dr. Ruben Hortensius (TSI Inc.) for technical support and guidance.

1. Fin fabrication
<ol>
	<li>Build a fin mold based on the desired shape design.
	<ol>
		<li>Design and build a custom 3D-printed gloss-finished mold of fin shape (Figure 1). See STL files for fabricating the mold in Supplementary Coding Files 1-4.</li>
		<li>Insert structural elements into the mold, such as a 3D-printed rigid plastic leading-edge spar. See the STL file of the spar in Supplementary Coding File 2.</li>
	</ol>
	</li>
	<li>Mi...

<ol>
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Planar laser-induced fluorescence is typically used to visualize aqueous flows by seeding the fluid with dye, which fluoresces when exposed to a laser sheet25,26. However, using PLIF to visualize deformations in compliant materials has not been previously reported, and this study describes an approach for obtaining time history measurements of high-resolution shape deformation in flexible solid fins using PLIF. Comparing these fin measurements with FSI simulation...

In nature, many fish species have evolved to use a variety of body and fin motions to achieve locomotion. Research to identify the principles of fish locomotion has helped drive the design of bioinspired propulsion systems, as biologists and engineers have worked together to develop capable next-generation propulsion and control mechanisms for underwater vehicles. Various research groups have studied fin configurations, shapes, materials, stroke parameters, and surface curvature control techniques1,2,3,4,5,6,7,8,9,10,11,12. The importance of characterizing tip vortex generation and wake inclination to understand thrust generation in single- and multi-fin systems has been documented in numerous studies, both computational and experimental13,14,15,16,17,18. For fin mechanisms made of compliant materials, shown in various studies to reduce wake inclination and increase thrust17, it is also essential to capture and accurately model their deformation time-history to pair with the flow structure analysis. These results can then be used to validate computational models, inform fin design and control, and facilitate active research areas in unsteady hydrodynamic loading on flexible materials, which need validation19. Studies have used direct high-speed image-based shape tracking in shark fins and other complex objects20,21,22, but the complex 3D fin shape often blocks optical access, making it difficult to measure. Thus, there is a pressing need for a simple and effective method to visualize flexible fin motion.
A material widely used in compliant fin mechanisms is polydimethylsiloxane (PDMS) due to its low cost, ease of use, ability to vary stiffness, and compatibility with underwater applications23, as described extensively in a review by Majidi et al.24. In addition to these benefits, PDMS is also optically transparent, which is conducive to measurements using an optical diagnostic technique such as planar laser-induced fluorescence (PLIF). Traditionally within experimental fluid mechanics25, PLIF has been used to visualize fluid flows by seeding the fluid with dye or suspended particles or taking advantage of quantum transitions from species already in the flow that fluoresce when exposed to a laser sheet26,27,28,29. This well-established technique has been used to study fundamental fluid dynamics, combustion, and ocean dynamics26,30,31,32,33.
In the present study, PLIF is used to obtain spatiotemporally resolved measurements of shape deformation in flexible fish-inspired robotic fins. Instead of seeding the fluid with dye, the underwater kinematics of a PDMS fin are visualized at various chordwise cross-sections. Although planar laser imaging can be performed on regular cast PDMS without additional fluorescence, modifying PDMS to enhance fluorescence can improve the signal-to-noise ratio (SNR) of the images by reducing the effects of background elements, such as the fin mounting hardware. PDMS can be made fluorescent by employing two methods, either by fluorescent particle seeding or pigmentation. It has been reported that, for a given part ratio, the former alters the stiffness of the resultant cast PDMS34. Therefore, a nontoxic, commercially available pigment was mixed with transparent PDMS to cast fluorescent fins for the PLIF experiments.
To provide an example of using these fin kinematics measurements for computational model validation, the experimental kinematics are then compared with values from the coupled fluid-structure interaction (FSI) models of the fin. The FSI models used in the computations are based on the first seven eigenmodes computed using the measured material properties for the fins. Successful comparisons validate fin models and provide confidence in using the computational results for fin design and control. Further, the PLIF results demonstrate that this method can be used to validate other numerical models in future studies. Additional information about these FSI models can be found in prior work35,36 and in fundamental texts of computational fluid dynamics methods37,38. Future studies can also allow for simultaneous measurements of solid deformations and fluid flows for improved experimental studies of FSI in robotic fins, bioinspired soft robots, and other applications. Furthermore, because PDMS and other compatible elastomers are widely used in various fields, including sensors and medical devices, visualizing deformations in flexible solids using this technique can benefit a larger community of researchers in engineering, physics, biology, and medicine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ADMET controller</td>
			<td>ADMET</td>
			<td>MTESTQuattro</td>
			<td></td>
		</tr>
		<tr>
			<td>Axon II</td>
			<td>Society of Robots</td>
			<td></td>
			<td>Microcontroller for the fin hardware</td>
		</tr>
		<tr>
			<td>Berkeley Nucleonics Delay Generator</td>
			<td>Berkeley Nucleonics Corp</td>
			<td>Model 525</td>
			<td>BNC delay generator and software</td>
		</tr>
		<tr>
			<td>BobCat Cam Config</td>
			<td>Imperx</td>
			<td></td>
			<td>Camera settings software</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Imperx</td>
			<td>B2340</td>
			<td>4 MegaPixel</td>
		</tr>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL Inc</td>
			<td></td>
			<td>Commercial structural dynamics software for fluid-structure interaction modeling</td>
		</tr>
		<tr>
			<td>D646WP Servo</td>
			<td>Hitec</td>
			<td>36646S</td>
			<td>32-Bit, Digital, High Torque, Waterproof Servo for the fin pitch rotation</td>
		</tr>
		<tr>
			<td>D840WP Servo</td>
			<td>Hitec</td>
			<td>36840S</td>
			<td>32-Bit, Multi Purpose, Waterproof, Steel Gear Servo for the fin stroke rotation</td>
		</tr>
		<tr>
			<td>Electric Pink fluorescent pigment</td>
			<td>Silc Pig</td>
			<td>PMS812C</td>
			<td></td>
		</tr>
		<tr>
			<td>EverGreen (532 nm dual pulsed Nd:YAG laser system)</td>
			<td>Quantel</td>
			<td>EVG00070</td>
			<td>Laser head and power supply, 70 mJ</td>
		</tr>
		<tr>
			<td>Force transducer</td>
			<td>ADMET</td>
			<td>SM-10-961</td>
			<td>10 lbf load cell</td>
		</tr>
		<tr>
			<td>FrameLink Express</td>
			<td>Imperx</td>
			<td></td>
			<td>Camera capture software</td>
		</tr>
		<tr>
			<td>Longpass fluorescence filter</td>
			<td>Edmund Optics</td>
			<td></td>
			<td>560 nm</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Software for image analysis</td>
		</tr>
		<tr>
			<td>Planetary centrifugal mixer</td>
			<td>THINKY MIXER</td>
			<td>AR-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber compounds</td>
			<td>Momentive</td>
			<td>RTV615</td>
			<td>Clear PDMS</td>
		</tr>
		<tr>
			<td>Stratasys J750</td>
			<td>Stratasys</td>
			<td></td>
			<td>3D printer, polyjet</td>
		</tr>
		<tr>
			<td>Universal testing machine</td>
			<td>ADMET</td>
			<td>eXpert 2611</td>
			<td>Table top model</td>
		</tr>
		<tr>
			<td>VeroBlack</td>
			<td>Stratasys</td>
			<td></td>
			<td>3D printer material to build the molds</td>
		</tr>
		<tr>
			<td>VeroGray</td>
			<td>Stratasys</td>
			<td></td>
			<td>3D printer material to build the molds</td>
		</tr>
	</tbody>
</table>

flapping soft fin deformation modeling using planar laser-induced fluorescence imaging

Propulsive mechanisms inspired by the fins of various fish species have been increasingly researched, given their potential for improved maneuvering and stealth capabilities in unmanned vehicle systems. Soft materials used in the membranes of these fin mechanisms have proven effective at increasing thrust and efficiency compared with more rigid structures, but it is essential to measure and model the deformations in these soft membranes accurately. This study presents a workflow for characterizing the time-dependent shape deformation of flexible underwater flapping fins using planar laser-induced fluorescence (PLIF). Pigmented polydimethylsiloxane fin membranes with varying stiffnesses (0.38 MPa and 0.82 MPa) are fabricated and mounted to an assembly for actuation in two degrees of freedom: pitch and roll. PLIF images are acquired across a range of spanwise planes, processed to obtain fin deformation profiles, and combined to reconstruct time-varying 3D deformed fin shapes. The data are then used to provide high-fidelity validation for fluid-structure interaction simulations and improve the understanding of the performance of these complex propulsion systems.

A trapezoidal fish-inspired artificial pectoral fin was cast in two different materials (PDMS 10:1 and 20:1, both mixed with fluorescent dye) out of a mold, each with a rigid leading-edge spar inserted into the leading quarter chord (Figure 2 and Figure 3). Tensile testing of the two fin materials (Figure 3) yielded elastic moduli of 0.38 MPa and 0.82 MPa for the PDMS 20:1 and PDMS 10:1 fins, respectively, with an R2 of 0...

Watch this Scientific Journal Video about Flapping Soft Fin Deformation Modeling using Planar Laser-Induced Fluorescence Imaging at JoVE.com

Flapping Soft Fin Deformation Modeling using Planar Laser-Induced Fluorescence Imaging

The present protocol involves the measurement and characterization of 3D shape deformation in underwater flapping fins built with polydimethylsiloxane (PDMS) materials. Accurate reconstruction of these deformations is essential for understanding the propulsive performance of compliant flapping fins.

Propulsive mechanisms inspired by the fins of various fish species have been increasingly researched, given their potential for improved maneuvering and ...

The ability to measure deformations of soft robotic fins non-intrusively is important because we can better inform fin design and control for underwater vehicles and validate computational models. We can repurpose an existing tool within fluid dynamics, called planer laser induced fluorescence and expand it for solids, which could then be used for simultaneous measurements of solids and fluids. This method is generalizable to soft robotic systems and materials.We can use this technique, not only to validate fluid structure interactions, but also to study flexible materials for sensors and medical applications. Begin by designing and building a custom 3D printed gloss finished mold of fin shape. Prepare an experimental setup by mounting a pulsed laser system on a rectangular glass water tank to generate a planer light sheet intersecting the tank at its midplane at 30 Hertz.Install a four megapixel charge coupled device, or CCD camera, with a 35 millimeter lens and a long pass fluorescence filter of 516 nanometers. After installation, perform a calibration of the micrometer to pixel conversion by taking a single image from the CCD camera with a ruler placed in the laser sheet plane. Then select two positions on the camera and divide the distance in micrometers by separating pixels.Ensure the micrometer to pixel ratio is small enough or in the sub millimeter range for the application. Synchronize the laser pulses and camera images with the flapping fin using trigger outputs from the fin software and signals from a delay generator and associated software. Ensure that all laser safety is per the institutional guidelines.To set the laser system, turn the laser system on with the power key that runs the chiller to cool the laser heads. The fault lights blink until the system is ready to power the lasers. Set the trigger source to external lamp, external Q switch.For both laser heads, set the laser energy to 60 to 80%of the full power and press each Q switch button. Then turn the lasers on by pressing the power button. Next, plug in the power cables to the camera and ensure proper connections to the computer before opening the camera setting software and selecting the proper port.After turning the delay generator on, connect the external gate channel to the fin trigger, channel E to the camera, and channels A to D to the laser. Then, open the delay generator software to select the pulse mode to burst and system resolution to four nanoseconds. Set the period in seconds.Adjust the external trigger/gate mode to triggered, threshold to 0.20 volts and trigger edge as rising. Also, set channels as described in the script. Align the fin, so the laser sheet passes through one cord wise section of the fin at a span wise position, and then secure the fin platform with the mounting hardware.Connect the power to the fin control hardware and fin motors to begin fin flapping with the selected kinematics, and turn off all the ambient lights. In the delay generator software, press run to begin the synchronized experiments and acquire images of the intersection of the laser sheet with the fin throughout the stroke cycle. Observe the fin flapping in the tank with laser sheet on and ambient lights off.When done, press stop before disconnecting the fin from the power source. Move the fin platform, so the laser sheet crosses at a new span wise position. Replace the fin with additional desired fin membranes and perform experiments as demonstrated before to acquire the images for the desired number of measurements.Analyze the image by extracting all the white objects representing fin cross sections from the BW area, filt dot M binary image and displaying the image with the M show dot M.Then create a trace of the binary image boundary for each image to obtain a 2D shape, by selecting all the fin pixels that touch the black background pixels. Compare the resulting fin shapes with the 3D fluid structure interaction, or FSI models, generated from the center lines, to showcase how the process can be used as high fidelity validation. The programmed fin kinematics yielded a stroke amplitude of 43 degrees and a pitch amplitude of 17 degrees.The image illustrates the comparisons at two positions in the stroke. One in the middle of the upstroke and one in the middle of the downstroke. Furthermore, the comparisons were made between the shape deformations of the PDMS 10 to one, and PDMS 20 to one fin.The 3D fin shapes were reconstructed from the planer laser induced fluorescence, FSI, and rigid cases in the mid upstroke, demonstrating the capability of the present technique for providing high fidelity validation for the FSI simulations. In the experiment, the fin cross section was not visible at every step due to the opaque rigid spar. The image shows the result where the fin was not visible.The most important thing to remember is to test the synchronization of the components before running full experiments. Once the timing is set, perform a test run first.

flapping-soft-fin-deformation-modeling-using-planar-laser-induced

Research

JoVE Journal

Bioengineering

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...