Z.C. acknowledges the support from Dartmouth startup fund and the Branco Weiss- Society for Science fellowship, administered by ETH Zurich. The authors thank Drs. Larry A. Taber, Benjamen A. Filas, Qiaohang Guo, and Yunfei Shi for helpful discussions, as well as the anonymous reviewers for comments.&#160;This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1313911. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.&#160;

1. Preparation of Tissue Culture Media
<ol>
	<li>Use a 0.5 L bottle of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 4.5 g/L glucose, sodium bicarbonate, and L-glutamine as the base for the culture media.</li>
	<li>In a sterile laminar flow hood, add 10 mL of antibiotics to the 0.5 L of DMEM.</li>
	<li>Using a sterile pipette, transfer 50 mL of the DMEM antibiotics solution to a sterile 50 mL conical tube.</li>
	<li>Add 50 mL of chick serum to the remaining DMEM antibiotics solution in the 0.5 L bottle in the sterile hood.</li>
	<li>Store the final solution (referred to hereafter as chick culture media [CCM]) in 50 mL conical tube aliquots at -20 &#176;C.</li>
</ol>
2. Egg Incubation
<ol>
	<li>Use delicate wipes with 70% ethanol to clean fertilized specific pathogen-free White Leghorn chicken eggs. Arrange eggs in a longitudinal orientation on holders.</li>
	<li>Turn on an egg incubator to set a target temperature of 37.5 &#176;C and maintain the humidity at 48 - 55%.&#160;The humidity is controlled by adding an appropriate amount of water to the incubator.</li>
	<li>Incubate the eggs to HH11-13, approximately 40 - 44 h.</li>
	<li>Let the eggs cool down at room temperature for approximately 15 - 30 min prior to spraying/cleaning with 70% ethanol.</li>
</ol>
3. Pull Glass Capillaries
<ol>
	<li>Mount a 10 cm-long glass capillary tubes with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm on a micropipette puller.</li>
	<li>Set the heat and pull parameters to 750 and 400, respectively. Press the pull button to pull the capillary tube into thin needles.</li>
</ol>
4. Filter Paper Carrier Method
<ol>
	<li>Cut circles of approximately 3 cm in diameter from filter paper.</li>
	<li>Cut a rectangle roughly 1 cm by 2 cm from the circle using a hole puncher. Make sure to remove any protruding or sharp corners.</li>
</ol>
5. Embryo Harvesting and Preparation
<ol>
	<li>Crack eggs from the bottom, pull apart the shell gently and carefully deposit contents into a 150 mm x 15 mm&#160;petri dish. To ensure the embryo side is up, keep the eggs in the same orientation they were incubated while cracking them.</li>
	<li>Remove the thin albumin using a disposable Pasteur pipette.</li>
	<li>Separate the thick albumin from the yolk using blunt ended forceps.&#160;Ensure that the thick albumin has been removed by lightly scraping the top of the yolk with the blunt ended forceps.</li>
	<li>Use fine-tipped forceps to center and&#160;place a filter paper ring over the embryo, matching the long axis of the ring with the long axis of the embryo.</li>
	<li>Cut the yolk surrounding the filter paper ring with scissors.</li>
	<li>Pull the ring and embryo off the yolk in an oblique direction towards the site where the yolk was first cut.</li>
	<li>Rinse the embryos in two sequential 100-mm dishes with room temperature 1x phosphate buffered saline (PBS).</li>
	<li>Place a filter paper ring into a 35-mm petri dish first. Then place the embryo dorsal side up onto the filter paper already in 35-mm petri dish.</li>
	<li>Place a stainless-steel ring on top of the filter paper sandwich. Make sure not to damage the embryo.</li>
	<li>Add 3&#160;mL&#160;of the previously prepared CCM to each petri dish.</li>
	<li>Remove the VM of each embryo by lightly skimming the pulled capillary needle across the top of the embryo and peeling the VM away, starting from the anterior end (right above the forebrain) and proceeding to the&#160;notochord (Figure 1A).</li>
	<li>Place eight 35-mm Petri dishes into one 150 mm dish that was lined with water saturated delicate task wipes (to maintain humidity).</li>
	<li>Place the 150-mm petri dish into a sealable plastic bag then fill the bag with a gas mixture comprised of 95% O2 and 5% CO2.</li>
	<li>Seal the bag and place it in a 37.5 &#176;C incubator.</li>
	<li>Incubate the embryos for an additional 27 h until HH15-HH16 (Figure 1B).</li>
</ol>
6. Inducing Surface Tension
<ol>
	<li>Remove the embryos from the incubator and use an optical coherence tomography (OCT) system to image them. Use OCT to determine the torsional angle of the neural tube (NT) (Figure 2).</li>
	<li>Transfer the embryos to a light microscope and visualize at 10X magnification. Use a 200 microliter pipette to incrementally remove 0.2 mL of media from the petri dish.</li>
	<li>Take brightfield images at each interval to observe the effects of the media-air interface on the embryo.</li>
	<li>Remove media until the surface tension across the embryo induces torsion (Figure 1C).</li>
	<li>Image the embryo using the OCT system once again to establish a final torsional angle for comparison to control embryos.&#160; NOTE: The Bright-field images were acquired by using a dissecting microscope. An optical coherence tomography system with an attached microscope was used to acquire cross-sectional image stacks of live embryos. Images were obtained in a 3 x 10 mm x 3 mm3 scanning domain, then processed in an imaging software. Lastly, the physical model images were taken with a digital single-lens reflex camera.</li>
</ol>
7. Physical Modeling of Surface Tension/VM Forces
<ol>
	<li>Develop a simplified 3D geometry that resembles an embryo between HH14-17 in commercial modeling software (Figure 3A).</li>
	<li>Design the negative mold of the 3D geometry in commercial 3D computer graphics software.</li>
	<li>Use a 3D printer loaded with 1.75 mm acrylonitrile butadiene styrene filament to 3D print the designed mold, in stereolithographic (.stl) format.</li>
	<li>To cast the mold, mix silicone rubber elastomer components A and B in equal parts and pour the mix into the mold promptly; set the casting mold to cure at room temperature for 12 h (Figure 3B).</li>
	<li>Mark the physical model of the embryo along the NT on the dorsal side to visualize torsion.</li>
	<li>Use a coverslip to replicate the force applied to the 3D physical model which mimics&#160;that of the VM or surface tension (Figure 3D).</li>
	<li>Insert a series of rigid wires of equal length to the side of the physical model. After the coverslip exerts an external force on the brain model, the wires become tilted at an angle that depends on the location. Determine the rotational angle by arctan of the projected length over the original length of each wire (Figure 3E).</li>
</ol>
8. Altering the Direction of the Heart Loop
<ol>
	<li>Follow the steps in 3.1 and 3.2 to obtain a pulled capillary tube.</li>
	<li>Follow the steps in 5.1 through 5.10 to prepare the embryo.</li>
	<li>Use a pair of forceps to flip the filter paper so that the embryo becomes ventral-side-up.</li>
	<li>Use the pulled capillary tube to cut&#160;open a slit in the splanchnopleure (SPL) membrane.</li>
	<li>Use the capillary tube to exert a mechanical force to push the heart from the right-hand side to the left-hand side.</li>
	<li>Follow the steps in 5.12 through 5.15 to observe the change in torsion.</li>
</ol>

<ol>
	<li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+Growth,+Remodeling,+and+Morphogenesis.">Biomechanics of Growth, Remodeling, and Morphogenesis.</a> Appl. Mech. Rev. 48, 487-545 (1995).</li><li>Wyczalkowski, M. A., Chen, Z., Filas, B. A., Varner, V. D., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+models+for+mechanics+of+morphogenesis.">Computational models for mechanics of morphogenesis.</a> Birth Defects Research Part C - Embryo Today: Reviews. 96, 132-152 (2012).</li><li>Savin, T., 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=On+the+growth+and+form+of+the+gut.">On the growth and form of the gut.</a> Nature. 476, 57-62 (2011).</li><li>Gjorevski, N., Nelson, C. 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+mechanics+of+development:+Models+and+methods+for+tissue+morphogenesis.">The mechanics of development: Models and methods for tissue morphogenesis.</a> Birth Defects Research Part C - Embryo Today: Reviews. 90, 193-202 (2010).</li><li>Shyer, A. E., 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=Villification:+how+the+gut+gets+its+villi.">Villification: how the gut gets its villi.</a> Science. 342, 212-218 (2013).</li><li>Ambrosi, D., 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=Perspectives+on+biological+growth+and+remodeling.">Perspectives on biological growth and remodeling.</a> Journal of the Mechanics and Physics of Solids. 59, 863-883 (2011).</li><li>Chen, Z., Majidi, C., Srolovitz, D. J., Haataja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+helical+ribbons.">Tunable helical ribbons.</a> Appl. Phys. Lett. 98, (2011).</li><li>Gerbode, S. J., Puzey, J. R., McCormick, A. G., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+cucumber+tendril+coils+and+overwinds.">How the cucumber tendril coils and overwinds.</a> Science. 337, 1087-1091 (2012).</li><li>Armon, S., Efrati, E., Kupferman, R., Sharon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+and+mechanics+in+the+opening+of+chiral+seed+pods.">Geometry and mechanics in the opening of chiral seed pods.</a> Science. 333, 1726-1730 (2011).</li><li>Filas, B. A., 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=A+potential+role+for+differential+contractility+in+early+brain+development+and+evolution.">A potential role for differential contractility in early brain development and evolution.</a> Biomech. Model. Mechanobiol. 11, 1251-1262 (2012).</li><li>Xu, 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=Axons+pull+on+the+brain,+but+tension+does+not+drive+cortical+folding.">Axons pull on the brain, but tension does not drive cortical folding.</a> J. Biomech. Eng. 132, 71013 (2010).</li><li>Chen, Z., Guo, Q., Dai, E., Forsch, N., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+embryonic+chick+brain+twists.">How the embryonic chick brain twists.</a> J. R. Soc. Interface. 13, (2016).</li><li>Manca, A., 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=Nerve+growth+factor+regulates+axial+rotation+during+early+stages+of+chick+embryo+development.">Nerve growth factor regulates axial rotation during early stages of chick embryo development.</a> Proc Natl Acad Sci U S A. 109, 2009-2014 (2012).</li><li>Shi, Y., Yao, J., Xu, G., Taber, L. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bending+of+the+looping+heart:+differential+growth+revisited.">Bending of the looping heart: differential growth revisited.</a> J. Biomech. Eng. 136, 1-15 (2014).</li><li>Shi, Y., 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=Bending+and+twisting+the+embryonic+heart:+A+computational+model+for+c-looping+based+on+realistic+geometry.">Bending and twisting the embryonic heart: A computational model for c-looping based on realistic geometry.</a> Front. Physiol. 5, (2014).</li><li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphomechanics:+Transforming+tubes+into+organs.">Morphomechanics: Transforming tubes into organs.</a> Current Opinion in Genetics and Development. 27, 7-13 (2014).</li><li>Hosseini, H. S., Beebe, D. C., Taber, L. 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+effects+of+the+surface+ectoderm+on+optic+vesicle+morphogenesis+in+the+chick+embryo.">Mechanical effects of the surface ectoderm on optic vesicle morphogenesis in the chick embryo.</a> J. Biomech. 47, 3837-3846 (2014).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev. Dyn. 88, 49-92 (1951).</li><li>Shi, Y., Varner, V. D., Taber, L. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+cytoskeletal+contraction+required+for+cardiac+fusion+before+but+not+after+looping+begins?.">Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?.</a> Phys. Biol. 12, 16012 (2015).</li><li>Garcia, K. E., Okamoto, R. J., Bayly, P. V., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraction+and+stress-dependent+growth+shape+the+forebrain+of+the+early+chicken+embryo.">Contraction and stress-dependent growth shape the forebrain of the early chicken embryo.</a> J. Mech. Behav. Biomed. Mater. 65, 383-397 (2017).</li><li>Faisst, A. M., Alvarez-Bolado, G., Treichel, D., Gruss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotatin+is+a+novel+gene+required+for+axial+rotation+and+left-right+specification+in+mouse+embryos.">Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos.</a> Mech. Dev. 113, 15-28 (2002).</li><li>Yalcin, H. C., Shekhar, A., Rane, A. a., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Dev. Dyn. 220, 284-289 (2001).</li><li>Camp&#224;s, O., 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=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11, 183-189 (2014).</li><li>Schierenberg, E., Junkersdorf, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+eggshell+and+underlying+vitelline+membrane+for+normal+pattern+formation+in+the+early+C.+elegans+embryo.">The role of eggshell and underlying vitelline membrane for normal pattern formation in the early C. elegans embryo.</a> Roux's Arch. Dev. Biol. 202, 10-16 (1992).</li><li>Chuai, M., Weijer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanisms+Underlying+Primitive+Streak+Formation+in+the+Chick+Embryo.">The Mechanisms Underlying Primitive Streak Formation in the Chick Embryo.</a> Current Topics in Developmental Biology. 81, 135-156 (2008).</li><li>Voronov, D. A., Alford, P. W., Xu, G., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanical+forces+in+dextral+rotation+during+cardiac+looping+in+the+chick+embryo.">The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo.</a> Dev. Biol. 272, 339-350 (2004).</li><li>Raya, A., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+the+establishment+of+left-right+asymmetry+in+the+chick+embryo.">Unveiling the establishment of left-right asymmetry in the chick embryo.</a> Mechanisms of Development. 121, 1043-1054 (2004).</li><li>Voronov, D. A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+looping+in+experimental+conditions:+Effects+of+extraembryonic+forces.">Cardiac looping in experimental conditions: Effects of extraembryonic forces.</a> Dev. Dyn. 224, 413-421 (2002).</li><li>Chen, Z., Huang, G., Trase, I., Han, X., Mei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Self-Assembly+of+a+Strain-Engineered+Flexible+Layer:+Wrinkling,+Rolling,+and+Twisting.">Mechanical Self-Assembly of a Strain-Engineered Flexible Layer: Wrinkling, Rolling, and Twisting.</a> Phys. Rev. Appl. 5, (2016).</li><li>Manner, J., Seidl, W., Steding, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+cervical+flexure:+an+experimental+study+on+chick+embryos.">Formation of the cervical flexure: an experimental study on chick embryos.</a> Acta Anat. (Basel). 152, 1-10 (1995).</li><li>Ware, M., Schubert, F. R. <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+the+early+axon+scaffold+in+the+rostral+brain+of+the+chick+embryo.">Development of the early axon scaffold in the rostral brain of the chick embryo.</a> J. Anat. 219, 203-216 (2011).</li><li>Ramasubramanian, A., 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=On+the+role+of+intrinsic+and+extrinsic+forces+in+early+cardiac+S-looping.">On the role of intrinsic and extrinsic forces in early cardiac S-looping.</a> Dev. Dyn. 242, 801-816 (2013).</li><li>Hoyle, C., Brown, N. a., Wolpert, L. <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+left/right+handedness+in+the+chick+heart.">Development of left/right handedness in the chick heart.</a> Development. 115, 1071-1078 (1992).</li><li>Nerurkar, N. L., Ramasubramanian, A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+adaptation+of+the+looping+embryonic+heart+to+altered+mechanical+loads.">Morphogenetic adaptation of the looping embryonic heart to altered mechanical loads.</a> Dev. Dyn. 235, 1822-1829 (2006).</li><li>Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+and+the+chick+embryo.">Left-right asymmetry and the chick embryo.</a> Semin. Cell Dev. Biol. 9, 67-76 (1998).</li><li>Roebroek, A. J., 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=Failure+of+ventral+closure+and+axial+rotation+in+embryos+lacking+the+proprotein+convertase+Furin.">Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.</a> Development. 125, 4863-4876 (1998).</li><li>Peebles, D. M., 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=Magnetic+resonance+proton+spectroscopy+and+diffusion+weighted+imaging+of+chick+embryo+brain+in+ovo.">Magnetic resonance proton spectroscopy and diffusion weighted imaging of chick embryo brain in ovo.</a> Dev. Brain Res. 141, 101-107 (2003).</li><li>Zhu, 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=Cerberus+regulates+left-right+asymmetry+of+the+embryonic+head+and+heart.">Cerberus regulates left-right asymmetry of the embryonic head and heart.</a> Curr. Biol. 9, 931-938 (1999).</li></ol>

The authors declare no conflicts of interest.

While physical phenomena play an integral role in morphogenesis26,27,28,29,30, the specific mechanical mechanisms, along with the coordination of mechanical and molecular mechanisms, remain largely unexplored. It is known that the ventral flexure and rightward torsion of the primitive brain are two central processes that contribute to early embryonic morphogen...

Modern developmental biology research largely focuses on understanding development from the perspective of molecular genetics1,2,3,4,5,6,7,8,9,10,11,12,13. It is known that physical phenomena play a central role in morphogenesis, or the generation of biological form14,15,16,17; however, specific mechanical mechanisms of development remain largely unstudied. Ventral flexure and rightward torsion of the primitive brain tube after Hamburger-Hamilton stage 11 (HH 11)18 are the two main processes that contribute to embryonic shape change19,20. In particular, the physical mechanism underlying the torsional development in the embryonic brain remains incompletely understood.
The embryonic torsion in chick embryo is among the earliest morphogenetic events of left-right (L-R) asymmetry in development. When the process of L-R asymmetry is perturbed, birth defects such as situs inversus, isomerism, or heterotaxia will occur21. 
Here we present a protocol which combines ex-ovo experiments22,23 with physical modeling to characterize mechanical forces during early embryonic brain development. The goal of the method presented is to identify the mechanical forces responsible for brain torsion and the factors that affect the degree of torsion during early development12. Based on the experimental observation that the vitelline membrane (VM) constrains the dorsal side of the embryo, we hypothesized that the VM provides the force necessary to induce torsion of the developing brain. Therefore, in this method, we removed the part of the VM that covers the brain area to find out the effects on brain torsion. Furthermore, the method of applying fluid surface tension was used to confirm the mechanical role of the VM and provide an estimate of the force needed for brain torsion, which had not been done previously. Measuring the forces during embryonic morphogenesis is a challenging task. Notably, in a pioneering study, Camp&#224;s and co-workers24 developed a novel method to quantify the cellular stresses using injected microdroplets. Nevertheless, this method was limited to measure forces at the cellular level, hence not applicable to probe forces at tissue- or organism-level. The protocol presented in this paper was developed to partially fill this gap.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Fertilized Specific pathogen-free White Leghorn chicken eggs</td>
			<td style="width:177px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:288px;">Optical Coherent Tomography Microscope</td>
			<td style="width:177px;">Thorlabs</td>
			<td style="width:119px;">GAN220C1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:288px;">Silicone elastomer</td>
			<td style="width:177px;">Smooth-On, Inc.</td>
			<td style="width:119px;">EcoFlex 00-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:119px;">MZ8</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:288px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Lonza</td>
			<td style="width:119px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Antibiotics</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">P4083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Chick serum</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">C5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Micropipette puller</td>
			<td style="width:177px;">Sutter Instrument</td>
			<td style="width:119px;">Model P-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Filter paper</td>
			<td style="width:177px;">Whatman</td>
			<td style="width:119px;">5202-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Phosphate buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Comsol MultiPhysics</td>
			<td style="width:177px;">Comsol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">3D computer graphics software</td>
			<td style="width:177px;">Rhino 5</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Microscope attached with OCT</td>
			<td style="width:177px;">Nikon&#160;</td>
			<td style="width:119px;">FN1</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:288px;">Digital single-lens reflex camera</td>
			<td style="width:177px;">EOS&#160;</td>
			<td style="width:119px;">Rebel T3i</td>
			<td></td>
		</tr>
	</tbody>
</table>

probing the roles of physical forces in early chick embryonic morphogenesis

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis is becoming increasingly recognized. In particular, the embryonic chick heart and brain tube, which undergo drastic morphological changes as they develop, are among the prime candidates to study the role of physical forces in morphogenesis. Progressive ventral bending&#160;and rightward torsion of the tubular embryonic chick brain happen at the earliest stage of organ-level left-right asymmetry in chick embryonic development. The vitelline membrane (VM) constrains the dorsal side of the embryo and has been implicated in providing the force necessary to induce torsion of the developing brain. Here we present a combination of new ex-ovo experiments and physical modeling to identify the mechanics of brain torsion. At Hamburger-Hamilton stage 11, embryos are harvested and cultured ex ovo (in media). The VM is subsequently removed using a pulled capillary tube. By controlling the level of the fluid and subjecting the embryo to a fluid-air interface, the fluid surface tension of the media can be used to replace the mechanical role of the VM. Microsurgery experiments were also performed to alter the position of the heart to find the resultant change in the chirality of brain torsion. Results from this protocol illustrate the fundamental roles of mechanics in driving morphogenesis.

In this study, the VM of the embryo at HH11 was removed from the anterior end to the thoracic flexure. The embryos were imaged by an OCT system. At this stage, the torsion of brain tube has not started (Figure 1A). After being incubated to HH15-16, embryos with their VM removed exhibited reduced brain tube torsion, approximately 35 degrees (Figure 1B) compared to control embryos, which exhibit torsion of around 90 degrees. When t...

Watch this Scientific Journal Video about Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis at JoVE.com

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

probing-roles-physical-forces-early-chick-embryonic

Research

JoVE Journal

Bioengineering

7.2K Views.  Dartmouth College. Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

Video: Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...