This research was supported by funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R15HD093024 and R01HD104910A and NSF CAREER Award Number 1752513.

Biomechanical Analysis of Peripheral Nerves Using DLT-Calibrated Stereo-Imaging

<ol>
	<li>Bueno, F. R., Shah, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+tensile+loading+for+the+tissue+engineering+of+nerves.">Implications of tensile loading for the tissue engineering of nerves.</a> Tissue Engineering Part B: Reviews. 14 (3), 219-233 (2008).</li><li>Grewal, R., Xu, J., Sotereanos, D. G., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+properties+of+peripheral+nerves.">Biomechanical properties of peripheral nerves.</a> Hand Clinics. 12 (2), 195-204 (1996).</li><li>Papagiannis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+behavior+and+viscoelastic+properties+of+peripheral+nerves+subjected+to+tensile+stress:+common+injuries+and+current+repair+techniques.">Biomechanical behavior and viscoelastic properties of peripheral nerves subjected to tensile stress: common injuries and current repair techniques.</a> Critical Reviews in Physical and Rehabilitation Medicine. 32 (3), 155-168 (2020).</li><li>Castaldo, J., Ochoa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+injury+of+peripheral+nerves.+Fine+structure+and+dysfunction.">Mechanical injury of peripheral nerves. Fine structure and dysfunction.</a> Clinics in Plastic Surgery. 11 (1), 9-16 (1984).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+of+impaired+axoplasmic+transport+and+neurofilament+compaction+in+traumatically+injured+axon+at+various+strains+and+strain+rates.">Extent of impaired axoplasmic transport and neurofilament compaction in traumatically injured axon at various strains and strain rates.</a> Brain Injury. 31 (10), 1387-1395 (2017).</li><li>Singh, A., Kallakuri, S., Chen, C., Cavanaugh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+changes+in+nerve+roots+due+to+tension+at+various+strains+and+strain+rates:+an+in-vivo+study.">Structural and functional changes in nerve roots due to tension at various strains and strain rates: an in-vivo study.</a> Journal of Neurotrauma. 26 (4), 627-640 (2009).</li><li>Singh, A., Lu, Y., Chen, C., Kallakuri, S., Cavanaugh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+traumatic+axonal+injury+to+determine+the+effects+of+strain+and+displacement+rates.">A new model of traumatic axonal injury to determine the effects of strain and displacement rates.</a> Stapp Car Crash Journal. 50, 601 (2006).</li><li>Singh, A., Lu, Y., Chen, C., Cavanaugh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+spinal+nerve+roots+subjected+to+tension+at+different+strain+rates.">Mechanical properties of spinal nerve roots subjected to tension at different strain rates.</a> Journal of Biomechanics. 39 (9), 1669-1676 (2006).</li><li>Singh, A., Shaji, S., Delivoria-Papadopoulos, M., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+responses+of+neonatal+brachial+plexus+to+mechanical+stretch.">Biomechanical responses of neonatal brachial plexus to mechanical stretch.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 13 (01), e8-e14 (2018).</li><li>Zapa&#322;owicz, K., Radek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+human+brachial+plexus.">Mechanical properties of the human brachial plexus.</a> Neurologia I Neurochirurgia Polska. 34, 89-93 (2000).</li><li>Zapa&#322;owicz, K., Radek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annales Academiae Medicae Stetinensis. 51 (2), 11-14 (2005).</li><li>Zapa&#322;owicz, K., Radek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+brachial+plexus+lesions+after+experimental+traction:+a+cadaveric+study.">The distribution of brachial plexus lesions after experimental traction: a cadaveric study.</a> Journal of Neurosurgery: Spine. 29 (6), 704-710 (2018).</li><li>Kawai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+of+the+brachial+plexus+in+rabbits.">Stretching of the brachial plexus in rabbits.</a> Acta Orthopaedica Scandinavica. 60 (6), 635-638 (1989).</li><li>Marani, E., Van Leeuwen, J., Spoor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tensile+testing+machine+applied+in+the+study+of+human+nerve+rupture:+a+preliminary+study.">The tensile testing machine applied in the study of human nerve rupture: a preliminary study.</a> Clinical Neurology and Neurosurgery. 95, 33-35 (1993).</li><li>Lee, S. K., Wolfe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+injury+and+repair.">Peripheral nerve injury and repair.</a> JAAOS-Journal of the American Academy of Orthopaedic Surgeons. 8 (4), 243-252 (2000).</li><li>Rickett, T., Connell, S., Bastijanic, J., Hegde, S., Shi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+mechanical+evaluation+of+nerve+stretch+injury.">Functional and mechanical evaluation of nerve stretch injury.</a> Journal of Medical Systems. 35, 787-793 (2011).</li><li>Topp, K. S., Boyd, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+biomechanics+of+peripheral+nerves:+nerve+responses+to+physical+stresses+and+implications+for+physical+therapist+practice.">Structure and biomechanics of peripheral nerves: nerve responses to physical stresses and implications for physical therapist practice.</a> Physical Therapy. 86 (1), 92-109 (2006).</li><li>Lu, Y., Chen, C., Kallakuri, S., Patwardhan, A., Cavanaugh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vivo+method+to+investigate+biomechanical+and+neurophysiological+properties+of+spine+facet+joint+capsules.">Development of an in vivo method to investigate biomechanical and neurophysiological properties of spine facet joint capsules.</a> European Spine Journal. 14 (6), 565-572 (2005).</li><li>Kallakuri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+stretching+of+cervical+facet+joint+capsule+and+related+axonal+changes.">Tensile stretching of cervical facet joint capsule and related axonal changes.</a> European Spine Journal. 17 (4), 556-563 (2008).</li><li>Abdel-Aziz, Y. I., Karara, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+linear+transformation+from+comparator+coordinates+into+object+space+coordinates+in+close-range+photogrammetry.">Direct linear transformation from comparator coordinates into object space coordinates in close-range photogrammetry.</a> Photogrammetric Engineering &amp; Remote Sensing. 81 (2), 103-107 (2015).</li><li>Pourcelot, P., Audigi&#233;, F., Degueurce, C., Geiger, D., Denoix, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+synchronise+cameras+using+the+direct+linear+transformation+technique.">A method to synchronise cameras using the direct linear transformation technique.</a> Journal of Biomechanics. 33 (12), 1751-1754 (2000).</li><li>Hedrick, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+techniques+for+two-+and+three-dimensional+kinematic+measurements+of+biological+and+biomimetic+systems.">Software techniques for two- and three-dimensional kinematic measurements of biological and biomimetic systems.</a> Bioinspiration &amp; Biomimetics. 3 (3), 034001 (2008).</li><li>Chen, L., Armstrong, C. W., Raftopoulos, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+on+the+accuracy+of+three-dimensional+space+reconstruction+using+the+direct+linear+transformation+technique.">An investigation on the accuracy of three-dimensional space reconstruction using the direct linear transformation technique.</a> Journal of Biomechanics. 27 (4), 493-500 (1994).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+in+vivo+biomechanical+testing+on+brachial+plexus+in+neonatal+piglets.">Methods for in vivo biomechanical testing on brachial plexus in neonatal piglets.</a> Journal of Visualized Experiments. (154), e59860 (2019).</li><li>Black, J., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi+camera+image+tracking.">Multi camera image tracking.</a> Image and Vision Computing. 24 (11), 1256-1267 (2006).</li><li>Cardenas-Garcia, J. F., Yao, H. G., Zheng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+reconstruction+of+objects+using+stereo+imaging.">3D reconstruction of objects using stereo imaging.</a> Optics and Lasers in Engineering. 22 (3), 193-213 (1995).</li><li>Mahan, M. A., Yeoh, S., Monson, K., Light, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+stretch+injury+to+peripheral+nerves:+biomechanical+results.">Rapid stretch injury to peripheral nerves: biomechanical results.</a> Neurosurgery. 85 (1), E137-E144 (2019).</li><li>Rydevik, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+mechanical+and+histological+study+of+acute+stretching+on+rabbit+tibial+nerve.">An in vitro mechanical and histological study of acute stretching on rabbit tibial nerve.</a> Journal of Orthopaedic Research. 8 (5), 694-701 (1990).</li><li>Mahan, M. A., Warner, W. S., Yeoh, S., Light, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid-stretch+injury+to+peripheral+nerves:+implications+from+an+animal+model.">Rapid-stretch injury to peripheral nerves: implications from an animal model.</a> Journal of Neurosurgery. 133 (5), 1537-1547 (2019).</li><li>Singh, A., Ferry, D., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+clinical+simulation+based+training+in+biomedical+engineering+education.">Efficacy of clinical simulation based training in biomedical engineering education.</a> Journal of Biomechanical Engineering. 141 (12), 121011-121017 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

Studies reporting biomechanical properties of peripheral nerves (PNs) because of stretch injury vary, and that variation can be attributed to testing methodologies such as testing equipment and elongation analysis5,6,7,8,9,10,11,12,<sup class="xr...

Peripheral nerves (PNs) undergo stretch during development, growth, normal joint movement, injury, and surgery1. PNs display viscoelastic properties to protect the nerve during regular movements2,3 and maintain the structural health of its nerve fibers2. Because PN response to mechanical stretch has been shown to depend on the type of nerve fiber damage4, injuries to adjacent connective tissues2,4, and testing approaches (i.e., loading rate or direction)5,6,7,8,9,10,11,12,13,14, it is essential to distinguish the biomechanical responses of PNs during normal range of motion versus non-physiological range at both slow- and rapid-stretch rates. This can further the understanding of the PN injury mechanism in response to stretch and aid in timely and optimized intervention1,4,15,16. There has been a growing trend in physical therapy to evaluate and intervene based on the relationship between nerve physiology and biomechanics17. By understanding the differences in PN biomechanics at various applied loads, physical therapists can be better prepared to modify current interventions17.
Available biomechanical data of PNs in response to stretch remains variable and can be attributed to testing equipment and procedures and differences in elongation data analysis5,6,7,8,9,10,11,12,13,14,16. Furthermore, measuring three-dimensional (3D) in-situ nerve displacement remains poorly described in the currently available literature. Previous studies have used stereo-imaging techniques to maximize the accuracy of 3D reconstruction of tissue displacement of facet joint capsules18,19. The direct linear transformation (DLT) technique enables the conversion of two or more two-dimensional (2D) views to 3D real-world coordinates (i.e., in mm)20,21,22. DLT provides a high-accuracy calibration method for stereo-imaging camera systems because it enables precise reconstruction of 3D positions, accounting for lens distortion, camera parameters, and image coordinates, and permits flexibility in stereo-imaging camera setup20,21,22. Studies using DLT-calibrated stereo-imaging camera systems are typically used to study locomotion and gait analysis22,23. This protocol aims to offer a detailed methodology to determine the in-situ strain of PNs at varying degrees of stretch using a DLT-calibrated stereo-imaging camera system and an open-source tracking software22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Clear Acrylic Plexiglass Square Sheet</td>
			<td>W W Grainger Inc</td>
			<td>BULKPSACR9</td>
			<td>Construct three-dimensional control volume</td>
		</tr>
		<tr>
			<td>Stereo-imaging camera system - ZED Mini Stereo Camera</td>
			<td>StereoLabs Inc.</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Imaging Software - ZED SDK</td>
			<td>StereoLabs Inc.</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Maintence Software - CUDA 12</td>
			<td>StereoLabs Inc.</td>
			<td>N/A</td>
			<td>Download to run ZED SDK&#160;</td>
		</tr>
		<tr>
			<td>Camera stand - Cast Iron Triangular Support Stand with Rod</td>
			<td>Telrose VWR Choice</td>
			<td>76293-346</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>MicroSribe G2 Digitizer with Immersion Foot Pedal</td>
			<td>SUMMIT Technology Group</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Proramming Software - MATLAB&#160;</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>version 2019A or newer</td>
		</tr>
		<tr>
			<td>DLTcal5.m</td>
			<td>Hedrick lab</td>
			<td>N/A</td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>DLTdv7.m</td>
			<td>Hedrick lab</td>
			<td>N/A</td>
			<td>Open Source</td>
		</tr>
	</tbody>
</table>

direct linear transformation for the measurement of in-situ peripheral nerve strain during stretching

Peripheral nerves undergo physiological and non-physiological stretch during development, normal joint movement, injury, and more recently while undergoing surgical repair. Understanding the biomechanical response of peripheral nerves to stretch is critical to the understanding of their response to different loading conditions and thus, to optimizing treatment strategies and surgical interventions. This protocol describes in detail the calibration process of the stereo-imaging camera system via direct linear transformation and the tracking of the three-dimensional in-situ tissue displacement of peripheral nerves during stretch, obtained from three-dimensional coordinates of the video files captured by the calibrated stereo-imaging camera system.
From the obtained three-dimensional coordinates, the nerve length, change in the nerve length, and percent strain with respect to time can be calculated for a stretched peripheral nerve. Using a stereo-imaging camera system provides a non-invasive method for capturing three-dimensional displacements of peripheral nerves when stretched. Direct linear transformation enables three-dimensional reconstructions of peripheral nerve length during stretch to measure strain. Currently, no methodology exists to study the in-situ strain of stretched peripheral nerves using a stereo-imaging camera system calibrated via direct linear transformation. Capturing the in-situ strain of peripheral nerves when stretched can not only aid clinicians in understanding underlying injury mechanisms of nerve damage when overstretched but also help optimize treatment strategies that rely on stretch-induced interventions. The methodology described in the paper has the potential to enhance our understanding of peripheral nerve biomechanics in response to stretch to improve patient outcomes in the field of nerve injury management and rehabilitation.

Using the described methodology, various output files are obtained. The DLTdv7.m *_xyzpts.csv (Supplemental File 12) contains the (x, y, z) coordinates in millimeters of each tracked point at each time frame that is further used to calculate the length, change in length, and strain of the stretched PN. Representative length-time, change in length-time, and strain-time plots of a stretched PN are shown in Figure 10. The stretched PN had an insertion marker, four markers along...

Read this Scientific Article Protocol about Direct Linear Transformation for the Measurement of In-Situ Peripheral Nerve Strain During Stretching at JoVE.com

Stereo-Imaging System DLT Calibration to Capture 3D In Situ Displacements of Stretched Peripheral Nerves

This protocol implements a stereo-imaging camera system calibrated using direct linear transformation to capture three-dimensional in-situ displacements of stretched peripheral nerves. By capturing these displacements, strain induced at varying degrees of stretch can be determined informing the stretch injury thresholds that can advance the science of stretch-dependent nerve repair.

Direct Linear Transformation for the Measurement of In-Situ Peripheral Nerve Strain During Stretching

Peripheral nerves undergo physiological and non-physiological stretch during development, normal joint movement, injury, and more recently while ...

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300 Views.  Drexel University. This protocol implements a stereo-imaging camera system calibrated using direct linear transformation to capture three-dimensional in-situ displacements of stretched peripheral nerves. By capturing these displacements, strain induced at varying degrees of stretch can be determined informing the stretch injury thresholds that can advance the science of stretch-dependent nerve repair. 

Video: Stereo-Imaging System DLT Calibration to Capture 3D In Situ Displacements of Stretched Peripheral Nerves

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

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 ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is essential to determine the effect of high ground impact forces on lower-body bone deformation to better understand the mechanisms of bone stress injuries. Conventional strain gauge measurement has been used to study in vivo tibia deformation. This method is associated with limitations including invasiveness of the procedure, involvement of few human subjects, and limited strain data from small bone surface areas. The current study intends to introduce a novel approach to study tibia bone strain under high impact loading conditions. A subject-specific musculoskeletal model was created to represent a healthy male (19 years, 80 kg, 1,800 mm). A flexible finite element tibia model was created based on a computed tomography (CT) scan of the subject&#39;s right tibia. Laboratory motion capture was performed to obtain kinematics and ground reaction forces of drop-landings from different heights (26, 39, 52 cm). Multibody dynamic computer simulations combined with a modal analysis of the flexible tibia were performed to quantify tibia strain during drop-landings. Calculated tibia strain data were in good agreement with previous in vivo studies. It is evident that this non-invasive approach can be applied to study tibia bone strain during high impact activities for a large cohort, which will lead to a better understanding of injury mechanism of tibia stress fractures.

During landing, lower-body bones experience large mechanical loads and are deformed. It is essential to measure bone deformation to better understand the mechanisms of bone stress injuries associated with impacts. A novel approach integrating subject-specific musculoskeletal modeling and finite element analysis is used to measure tibial strain during dynamic movements.

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...