This work was supported by the startup fund provided by Iowa State University and by the National Institute of General Medical Sciences (R35GM128747).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC, 8-16-8333-I) of Iowa State University.
1. Synthesis of the integrative tension sensor
<ol>
	<li>Customize and order ssDNAs (see the Table of Materials). 
	NOTE: The ssDNA sequences are as follows. The upper strand is /5ThioMC6-D/GGG AGG ACG CAG CGG GCC/3BHQ_2/. The lower strands are as follows. 
	12 pN ITS: /5Cy3/GGC CCG CTG CGT CCT CCC /3Bio/ 
	23 pN ITS: /5Cy3/GGC CCG CTG CGT CC /iBiodT/ CCC 
	33 pN ITS: /5Cy3/GGC CCG CTG CG/iBiodT/ CCT CCC 
	43 pN ITS: /5Cy3/GGC CCG C/iBiodT/G CGT CCT CCC 
	54 pN ITS: /5Biosg//iCy3/GGC CCG CTG CGT CCT CCC 
	Here, /5ThioMC6-D/ represents a thiol modifier C6 S-S (disulfide) at the 5&#39; end. /3BHQ_2/ represents a Black Hole Quencher 2 at the 3&#39; end. /3Bio/, /5Biosg/, and /iBiodT/ are biotinylation at the 3&#39; end, the 5&#39; end, and on the thymine of internal DNA, respectively. /iCy3/ represents a Cy3 inserted into the internal backbone of ssDNA. These codes are used by the specific commercial supplier used here and may be different in other DNA companies.</li>
	<li>Conjugate RGD peptide to the SH-ssDNA-quencher. 
	NOTE: The reagents used are phosphate-buffered saline (PBS), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), RGD peptide, and tris-(2-carboxyethyl)phosphine hydrochloride, also known as TCEP-HCl.
	<ol>
		<li>Deprotect the thiol modifier on the upper strand DNA using TCEP solution. 
		NOTE: Thiol-modified DNAs ordered from a commercial source are shipped in oxidized form, with the sulfur atoms protected by a disulfide bond that needs be reduced prior to RGD-DNA conjugation. TCEP is an odorless reduction reagent used for the thiol deprotection.
		<ol>
			<li>Dissolve 14.3 mg of TCEP-HCl in 300 &#181;L of water. Adjust the pH of the TCEP solution to ~7.2&#8211;7.4 with 1 M NaOH solution (about 150 &#181;L), using pH test papers. Add pure water to make a final volume of 0.5 mL. The final TCEP concentration is 100 mM. 
			NOTE: It is recommended to make TCEP solution fresh every time for DNA thiol deprotection. Avoid stocking TCEP solution for a long time.</li>
			<li>Add 100 &#181;L of 0.5 M ethylenediaminetetraacetic acid (EDTA) solution and 400 &#181;L of water to the TCEP solution.</li>
			<li>Add 10 &#181;L of the TCEP+EDTA solution to 20 &#181;L of 1 mM thiol-DNA-BHQ2 in PBS. Let the solution react for 30 min at room temperature. 
			NOTE: The disulfide bond of 5&#39; thiol modifier C6 S-S conjugation at this ssDNA will be cleaved by TCEP, leaving the thiol group ready for reaction with maleimide in the next steps. The TCEP does not interfere with the following thiol-maleimide reaction; thus, it is not necessary to passivate TCEP after this step.</li>
		</ol>
		</li>
		<li>Conjugate RGD-NH2 with sulfo-SMCC.
		<ol>
			<li>Dissolve 5 mg of RGD-NH2 in 100 &#181;L of PBS to obtain 11 mM RGD-NH2.</li>
			<li>Add 200 &#181;L of pure water to the tube with 2 mg of sulfo-SMCC (premeasured by the supplier). Smash the sulfo-SMCC solid with a pipette tip and vigorously pipette at the same time to facilitate dissolving the SMCC in the water. The concentration of sulfo-SMCC will be 23 mM. 
			NOTE: Because the NHS ester in sulfo-SMCC is subject to hydrolysis and has a lifetime of a few minutes, this dissolving step should be completed within 1 min to avoid excessive loss of the NHS ester due to hydrolysis. Use pure water to dissolve sulfo-SMCC because its solubility in PBS or other buffers is poor.</li>
			<li>Add 40 &#181;L of sulfo-SMCC solution to the 100 &#181;L RGD-NH2 solution and incubate it for 20 min. 
			NOTE: The NHS ester in sulfo-SMCC will react with the amine group in RGD-NH2, forming an amide bond that links RGD and sulfo-SMCC.</li>
		</ol>
		</li>
		<li>Conjugate RGD-SMCC with the SH-ssDNA-quencher.
		<ol>
			<li>Mix the solutions prepared in steps 1.2.1 and 1.2.2 and incubate the mixture for 1 h at room temperature. Move the solution to a refrigerator set at 4 &#176;C and let it further react overnight. The thiol conjugated on the upper strand DNA will react with maleimide on sulfo-SMCC, forming a thioether group that links RGD with the upper strand DNA.</li>
		</ol>
		</li>
		<li>Perform ethanol precipitation to purify RGD-ssDNA-quencher from unreacted sulfo-SMCC, RGD, and TCEP.
		<ol>
			<li>Chill 10 mL of 100% ethanol in a 15 mL conical tube in a -20 &#176;C freezer for at least 30 min.</li>
			<li>Mix a 3 M NaCl solution, a DNA solution, and chilled ethanol at a volume ratio of 1:10:25. Keep the mixed liquid in a -20 &#176;C freezer for 30 min or until the DNA is precipitated.</li>
			<li>Centrifuge the liquid at 10,000 x g for 30 min. Discard the supernatant.</li>
			<li>Add PBS to the DNA pellet. Measure the DNA concentration using a spectrometer.</li>
		</ol>
		</li>
		<li>(Optional) Perform DNA electrophoresis to gain a higher RGD-ssDNA purity. 
		NOTE: With the above protocol, about 70%&#8211;90% of the ssDNA will be conjugated with RGD. If higher purity is desired, DNA electrophoresis can also be performed to separate conjugated DNA from unconjugated DNA.
		<ol>
			<li>Assemble a vertical electrophoresis system.</li>
			<li>Prepare a 10% polyacrylamide gel solution by mixing 50 mL of pure water, 20 mL of 40% acrylamide gel, 8 mL of 10x tris-borate-EDTA (TBE) buffer and 800 &#181;L of 10% ammonium persulfate (APS).</li>
			<li>Add 80 &#181;L of tetramethylethylenediamine (TEMED) to the gel solution. Pour the solution into the glass chamber of the electrophoresis system. Insert a single-well comb to create a well on top of the gel. Wait for 10 min till the gel is crosslinked.</li>
			<li>Mix the DNA solution with 100% glycerol at a volume ratio of 1:1. Remove the comb and load the DNA solution to the gel well.</li>
			<li>Run the gel at a voltage of 200 V. Observe two DNA bands in which the lagging one is the conjugated DNA.</li>
			<li>Cut the gel band with the conjugated DNA off and dice it into small pieces.</li>
			<li>Collect the diced gel in two 1.5 mL centrifuge tubes with filters. Add 500 &#181;L of PBS to each tube.</li>
			<li>Put the PBS-soaked gel in a dark place at room temperature for 1 day. DNA will diffuse out of the gel pieces into the PBS.</li>
			<li>Collect the DNA solution by centrifuging the tubes at 1,000 x g for 2 min.</li>
			<li>Perform another round of ethanol precipitation (step 1.2.4) to collect the DNA.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Synthesize the ITS by hybridizing RGD-ssDNA-quencher and dye-ssDNA-biotin.
	<ol>
		<li>Mix the solutions of the upper strand and the lower strand DNAs at a molar ratio of 1.1:1. Keep the mixture at 4 &#176;C overnight to produce the ITS by DNA hybridization. 
		NOTE: A molar ratio of 1.1:1 for DNA hybridization is used instead of 1:1. Excessive RGD-ssDNA-quencher is used to ensure that all dye-ssDNA-biotin will hybridize with RGD-ssDNA-quencher. RGD-ssDNA-quencher without hybridization will be washed off during surface coating, which will not affect the ITS surface coating quality.</li>
		<li>Aliquot and store the ITS solution at -20 &#176;C for long term storage. 
		NOTE: The storage buffer is PBS and the storage concentration should generally be above 10 &#181;M. For regular use, ITS solution can be stored at 4 &#176;C for a few weeks without noticeable quality deterioration.</li>
	</ol>
	</li>
</ol>
2. Preparation of ITS surfaces by immobilizing the ITS on glass-bottomed Petri dishes
NOTE: The reagents used are biotinylated bovine serum albumin (BSA-biotin), avidin protein, and ITS. Chill all reagents and PBS buffer to around 0 &#176;C on ice with an ice bucket.
<ol>
	<li>Load 200 &#181;L of 0.1 mg/mL BSA-biotin in PBS onto the well of a glass-bottomed Petri dish (29 mm in diameter, with a 14 mm well). Incubate it for 30 min at 4 &#176;C in a refrigerator. BSA-biotin is physically adsorbed on the glass surface.</li>
	<li>Suck out the solution from the well using a pipette and add 200 &#181;L of PBS back to the well to wash the surface. Repeat this 3x to complete the washing.</li>
	<li>Incubate 200 &#181;L of 50 &#181;g/mL avidin protein in the well for 30 min at 4 &#176;C. Wash it 3x with PBS. After this step, the avidin protein binds to the BSA-biotin and becomes immobilized on the glass surface.</li>
	<li>Incubate 200 &#181;L of 0.1 &#181;M ITS in the well for 30 min at 4 &#176;C. Wash the dish with 3x with PBS. Leave PBS in the well until the cell plating. After this step, the ITS with the biotin tag binds to the avidin protein and becomes immobilized on the surface. 
	NOTE: One precaution critical for ITS coating quality and homogeneity during the ITS immobilization is to never let the Petri dish surface dry up. Keeping all reagents and PBS at 4 &#176;C or around 0 &#176;C on ice with an ice bucket helps to reduce the water evaporation rate during the washing steps, when PBS is drawn out from the well.</li>
</ol>
3. Cell plating onto ITS surfaces
<ol>
	<li>Culture fish keratocytes. 
	NOTE: Here, goldfish (Carassius auratus) is used as an example, but other fish species may also work.
	<ol>
		<li>Pluck a piece of fish scale from a fish with a flat-tip tweezer. Gently press the scale onto the well of a glass-bottomed Petri dish, with the inner side of the scale contacting the glass. Wait for ~30 s for the fish scale to adhere well to the glass surface of the dish.</li>
		<li>Add 1 mL of Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM) complete medium to fully cover the fish scale. Keep the Petri dish in a dark and humid box for 6&#8211;48 h at room temperature. 
		NOTE: IMDM complete medium contains 79% IMDM + 20% fetal bovine serum (FBS) + 1% penicillin. No extra supply of oxygen or CO2 is needed. A few tissue paper rolls soaked with water can be left in the box to maintain high humidity in the box. Fish keratocytes will migrate out of the fish scale as a sheet of cells after a few hours. This can be observed with a tissue culture microscope. The cells are generally viable at 48 h.</li>
	</ol>
	</li>
	<li>Detach and plate the keratocytes cells on ITS surfaces.
	<ol>
		<li>Remove the medium from the Petri dish in which the fish scale was cultured. Wash the well 1x with cell detaching solution, and add detaching solution to cover the fish scale and incubate for 3 min. 
		NOTE: The recipe for the cell detaching EDTA solution is as follows: 100 mL of 10x Hanks&#39; Balanced Salt Solution (HBSS) + 10 mL of 1 M HEPES (pH 7.6) + 10 mL of 7.5% sodium bicarbonate + 2.4 mL of 500 mM EDTA +1 L of H2O. The pH is adjusted to 7.4.</li>
		<li>Suck out all cell detaching solution and add IMDM complete medium. Disperse the keratocytes by gentle pipetting. Adjust the cell solution density to the desired level (1 x 104&#8211;1 x 105/mL). Plate the cells on the ITS surface (prepared according to section 2). Incubate the cells at room temperature for 30 min before imaging. 
		NOTE: Cell counting can be done with a hemocytometer after detaching the cells with detaching solution. The cell plating density is relatively low so that the keratocytes have plenty of surface area to migrate.</li>
	</ol>
	</li>
	<li>Plate CHO-K1 cells and NIH 3T3 cells on ITS surfaces. 
	NOTE: The medium for CHO-K1 cells is 89% Ham&#8217;s F12 + 10% FBS + 1% penicillin. The medium for NIH 3T3 cells is 89% Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) + 10% calf bovine serum (CBS) + 1% penicillin. The culture conditions are 37 &#176;C and 5% CO2.
	<ol>
		<li>Culture CHO-K1 cells or NIH 3T3 cells to 80%&#8211;100% confluence in a Petri dish (or culture flask).</li>
		<li>Warm the cell detaching solution and culture medium to 37 &#176;C.</li>
		<li>Remove all culture medium in the Petri dish with a pipette. Wash the cells 1x with cell detaching solution. Cover the cells with detaching solution and incubate it at 37 &#176;C for 10 min.</li>
		<li>Disperse the cells by pipetting. Collect the cell solution and centrifuge it at 300 x g for 3 min.</li>
		<li>Suck out the supernatant and add complete medium or serum-free medium.</li>
		<li>Adjust the cell solution density to the desired level (1 x 105 &#8211; 1 x 106/mL). Plate the cells on the ITS surfaces (prepared according to section 2). Incubate the cells at 37 &#176;C for 1&#8211;2 h before imaging, or 30 min before video recording.</li>
	</ol>
	</li>
</ol>
4. Imaging, video recording, and real-time integrin tension mapping
<ol>
	<li>Quasi-real-time imaging of integrin tension in live cells
	<ol>
		<li>Incubate keratocytes on ITS surfaces (prepared according to section 2) for 30 min at room temperature, or incubate CHO-K1 cells or NIH 3T3 cells for 1 h on ITS surfaces in an incubator at 37 &#176;C.</li>
		<li>Mount the Petri dish on a fluorescence microscope stage. Perform cellular force imaging with an exposure time of 1 s. The magnification is 40x or 100x.</li>
		<li>Take a video of ITS images with a frame interval of 20 s for keratocytes, or 1&#8211;2 min for CHO-K1 cells or NIH 3T3 cells.</li>
		<li>Use MATLAB or another image analysis tool to subtract a previous frame of the ITS video from a current frame to compute the ITS signal newly produced in the latest frame interval. The newly produced ITS signal represents the integrin tension generated in the latest frame interval, thereby reporting the cellular force in a quasi-real-time manner.</li>
	</ol>
	</li>
	<li>Coimaging of integrin tension and cellular structure in immunostained cells
	<ol>
		<li>After step 3.2.2 or step 3.3.6, perform immunostaining to mark the target structural proteins. 
		NOTE: Use different dyes to avoid the fluorescence crosstalk between integrin tension imaging and cell structural imaging. In this manuscript, Cy5 fluorophore was used for phalloidin and Alexa 488 was used for vinculin staining. The concentration of both primary and secondary antibody is 2.5 &#181;g/mL. The concentration for phalloidin is 1.5 units/&#181;L.</li>
		<li>Perform multifluorescent channel imaging to acquire both an integrin tension map and cell structural imaging.</li>
	</ol>
	</li>
</ol>

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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-Path+Quenchers:+Efficient+Quenching+of+Common+Fluorophores.">Multi-Path Quenchers: Efficient Quenching of Common Fluorophores.</a> Bioconjugate Chemistry. 22 (11), 2345-2354 (2011).</li><li>Mondal, G., Barui, S., Chaudhuri, 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+relationship+between+the+cyclic-RGDfK+ligand+and+&#945;v&#946;3+integrin+receptor.">The relationship between the cyclic-RGDfK ligand and &#945;v&#946;3 integrin receptor.</a> Biomaterials. 34 (26), 6249-6260 (2013).</li><li>Wang, X., 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=Integrin+Molecular+Tension+within+Motile+Focal+Adhesions.">Integrin Molecular Tension within Motile Focal Adhesions.</a> Biophysical Journal. 109 (11), 2259-2267 (2015).</li><li>Euteneuer, U., Schliwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent,+directional+motility+of+cells+and+cytoplasmic+fragments+in+the+absence+of+microtubules.">Persistent, directional motility of cells and cytoplasmic fragments in the absence of microtubules.</a> Nature. 310 (5972), 58-61 (1984).</li><li>Kucik, D. F., Elson, E. L., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+does+not+produce+membrane+flow.">Cell migration does not produce membrane flow.</a> The Journal of Cell Biology. 111 (4), 1617-1622 (1990).</li><li>Mueller, 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=Load+Adaptation+of+Lamellipodial+Actin+Networks.">Load Adaptation of Lamellipodial Actin Networks.</a> Cell. , (2017).</li><li>Sarkar, A., Zhao, Y., Wang, Y., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-activatable+coating+enables+high-resolution+cellular+force+imaging+directly+on+regular+cell+culture+surfaces.">Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.</a> Physical Biology. 15 (6), 065002 (2018).</li><li>Mosayebi, M., Louis, A. A., Doye, J. P. K., Ouldridge, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-Induced+Rupture+of+a+DNA+Duplex:+From+Fundamentals+to+Force+Sensors.">Force-Induced Rupture of a DNA Duplex: From Fundamentals to Force Sensors.</a> ACS Nano. 9 (12), 11993-12003 (2015).</li><li>Bockelmann, U., Essevaz-Roulet, B., Heslot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Stick-Slip+Motion+Revealed+by+Opening+DNA+with+Piconewton+Forces.">Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces.</a> Physical Review Letters. 79 (22), 4489-4492 (1997).</li><li>Krautbauer, R., Rief, M., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unzipping+DNA+oligomers.">Unzipping DNA oligomers.</a> Nano Letters. 3 (4), 493-496 (2003).</li><li>de Gennes, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+pull+out+force+on+DNA+hybrids.">Maximum pull out force on DNA hybrids.</a> Comptes Rendus de l&#8217;Acad&#233;mie des Sciences - Series IV - Physics. 2 (10), 1505-1508 (2001).</li><li>Hatch, K., Danilowicz, C., Coljee, V., Prentiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+that+the+shear+force+required+to+separate+short+double-stranded+DNA+does+not+increase+significantly+with+sequence+length+for+sequences+longer+than+25+base+pairs.">Demonstration that the shear force required to separate short double-stranded DNA does not increase significantly with sequence length for sequences longer than 25 base pairs.</a> Physical Review E. 78 (1), 011920 (2008).</li></ol>

The authors have nothing to disclose.

The ITS is a highly accessible yet powerful technique for cellular force mapping in terms of both synthesis and application. With all materials ready, the ITS can be synthesized within 1 day. During experiments, only three steps of surface coating are needed prior to cell plating. Recently, we further simplified the coating procedure to one step by directly linking the ITS to bovine serum albumin, which enables direct physical adsorption of the ITS to glass or polystyrene surfaces33. The ITS bring...

Cells rely on integrins to adhere and exert cellular forces to extracellular matrix. Integrin-mediated cell adhesion and force transmission are crucial for cell spreading1,2, migration3,4, and survival5,6,7. In the long term, integrin biomechanical signaling also influences cell proliferation8,9,10 and differentiation11,12. Researchers have developed various methods to measure and map integrin-transmitted cellular forces at the cell-matrix interface. These methods are based on elastic substratum13, array of micropost14, or atomic force microscopy (AFM)15,16. Elastic substratum and micropost methods rely on the deformation of substrates to report the cellular stress and have limitations in terms of spatial resolution and force sensitivity. AFM has high force sensitivity, but it cannot detect force at multiple spots simultaneously, making it difficult to map cellular force transmitted by integrins.
In recent years, several techniques were developed to study cellular force at the molecular level. A collection of molecular tension sensors based on polyethylene glycol17,18, spider silk peptide19, and DNA20,21,22,23 were developed to visualize and monitor tension transmitted by molecular proteins. Among these techniques, DNA was first adopted as the synthesis material in the tension gauge tether (TGT), a rupturable linker that modulates the upper limit of integrin tensions in live cells22,24. Later, DNA and fluorescence resonance transfer technique were combined to create hairpin DNA-based fluorescent tension sensors first by Chen&#8217;s group23 and Salaita&#8217;s group20. The hairpin DNA-based tension sensor reports integrin tension in real-time and has been successfully applied to the study of a series of cellular functions21. Afterward, Wang&#8217;s lab combined a TGT with the fluorophore-quencher pair to report integrin tension. This sensor is named an ITS25,26. The ITS is based on double-stranded DNA (dsDNA) and has a broader dynamic range (10-60 pN) for integrin tension calibration. In contrast to hairpin DNA-based sensors, the ITS does not report cellular force in real-time but records all historic integrin events as the footprint of cellular force; this signal accumulation process improves the sensitivity for cellular force imaging, making it feasible to image cellular force even with a low-end fluorescence microscope. The synthesis of ITS is relatively more convenient as it is created by hybridizing two single-stranded DNAs (ssDNA).
The ITS is an 18-base-paired dsDNA conjugated with biotin, a fluorophore, a quencher (Black Hole Quencher 2 [BHQ2])27, and a cyclic arginylglycylaspartic acid (RGD) peptide28 as an integrin peptide ligand (Figure 1). The lower strand is conjugated with the fluorophore (Cy3 is used in this manuscript, while other dyes, such as Cy5 or Alexa series, have also been proven feasible in our lab) and the biotin tag, with which the ITS is immobilized on a substrate by biotin-avidin bond. The upper strand is conjugated with the RGD peptide and the Black Hole Quencher, which quenches Cy3 with approximately 98% quenching efficiency26,27. With the protocol presented in this paper, the coating density of the ITS on a substrate is around 1,100/&#181;m2. This is the density we previously calibrated for 18 bp biotinylated dsDNA coated on the neutrAvidin-functionalized substrate by following the same coating protocol29. When cells adhere to the substrate coated with the ITS, integrin binds the ITS through RGD and transmits tension to the ITS. The ITS has a specific tension tolerance (Ttol) which is defined as the tension threshold that mechanically separates the dsDNA of the ITS within 2 s22. ITS rupture by integrin tension leads to the separation of the quencher from the dye that subsequently emits fluorescence. As a result, the invisible integrin tension is converted to a fluorescence signal and the cellular force can be mapped by fluorescence imaging.
To demonstrate the application of the ITS, we use fish keratocyte here, a widely used cell model for cell migration study30,31,32, CHO-K1 cell, a commonly used nonmotile cell line, and NIH 3T3 fibroblast. Coimaging of integrin tension and cell structures is also conducted.

<table><tbody><tr><td>BSA-biotin</td><td>Sigma-Aldrich</td><td>A8549</td><td></td></tr><tr><td>Neutravidin</td><td>Thermo Fisher Scientific</td><td>31000</td><td></td></tr><tr><td>Streptavidin</td><td>Thermo Fisher Scientific</td><td>434301</td><td></td></tr><tr><td>upper strand DNA</td><td>Integrated DNA Technologies</td><td>N/A</td><td>Customer designed. DNA sequence is shown in PROTOCOL section</td></tr><tr><td>lower strand DNA</td><td>Integrated DNA Technologies</td><td>N/A</td><td>Customer designed. DNA sequences are shown in PROTOCOL section.</td></tr><tr><td>sulfo-SMCC</td><td>Thermo Fisher Scientific</td><td>A39268</td><td></td></tr><tr><td>Cyclic peptide RGD with an amine group</td><td>Peptides International</td><td>PCI-3696-PI</td><td></td></tr><tr><td>IMDM</td><td>ATCC</td><td>&amp;lrm;62996227</td><td></td></tr><tr><td>FBS</td><td>ATCC</td><td>302020</td><td></td></tr><tr><td>Penicillin</td><td>gibco</td><td>15140122</td><td></td></tr><tr><td>TCEP</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>200 uL petri dish</td><td>Cellvis</td><td>D29-14-1.5-N</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>N/A</td><td>spectrometer</td></tr><tr><td>SE410 Tall Air-Cooled Vertical Protein Electrophoresis Unit</td><td>Hoefer</td><td>SE410-15-1.5</td><td>Device for electroporesis</td></tr><tr><td>CHO-K1 cell line</td><td>ATCC</td><td>CCL-61</td><td></td></tr><tr><td>NIH/3T3 cell line</td><td>ATCC</td><td>CRL-1658</td><td></td></tr><tr><td>Anti-Vinculin Antibody</td><td>EMD Millipore</td><td>90227</td><td>Primary antibody for vinculin immunostaining</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A28175</td><td>Secondary antibody for vinculin immunostaining</td></tr><tr><td>Alexa Fluor 647 Phalloidin</td><td>Invitrogen</td><td>A22287</td><td></td></tr><tr><td>Eclipse Ti</td><td>Nikon</td><td>N/A</td><td>microscope</td></tr></tbody></table>

imaging integrin tension and cellular force at submicron resolution with an integrative tension sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10&#8211;60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

With the ITS, the integrin tension map of fish keratocytes was captured. It shows that a keratocyte migrates and generates integrin tension at two force tracks (Figure 2A). The resolution of the force map was calibrated to be 0.4 &#181;m (Figure 2B). High integrin tension concentrates at the rear margin (Figure 3A). The ITS also shows different specific patterns of different cells. A nonmotile cell, ...

Watch this Scientific Journal Video about Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor at JoVE.com

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...

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7.6K Views.  Iowa State University. Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

Video: Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

Biochemistry

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant adsorption rates, evaluating equilibrium surface tensions as a function of bulk surfactant concentration, and relating how surface tension changes with changes in the interfacial area following equilibration. Simultaneous visualization of the interface using fluorescence imaging with a high-speed confocal microscope allows the direct evaluation of structure-function relationships. In the capillary pressure microtensiometer (CPM), a hemispherical air bubble is pinned at the end of the capillary in a 1 mL volume liquid reservoir. The capillary pressure across the bubble interface is controlled via a commercial microfluidic flow controller that allows for model-based pressure, bubble curvature, or bubble area control based on the Laplace equation. Compared to previous techniques such as the Langmuir trough and pendant drop, the measurement and control precision and response time are greatly enhanced; capillary pressure variations can be applied and controlled in milliseconds. The dynamic response of the bubble interface is visualized via a second optical lens as the bubble expands and contracts. The bubble contour is fit to a circular profile to determine the bubble curvature radius, R, as well as any deviations from circularity that would invalidate the results. The Laplace equation is used to determine the dynamic surface tension of the interface. Following equilibration, small pressure oscillations can be imposed by the computer-controlled microfluidic pump to oscillate the bubble radius (frequencies of 0.001-100 cycles/min) to determine the dilatational modulus The overall dimensions of the system are sufficiently small that the microtensiometer fits under the lens of a high-speed confocal microscope allowing fluorescently tagged chemical species to be quantitatively tracked with submicron lateral resolution.

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant ...

Engineering

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Biology

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Developmental Biology

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Quantitative assessment of cellular forces and motion advanced considerably over the last four decades. These advancements provided the framework to examine insightful mechanical signaling processes in cell culture systems. However, the field currently faces three problems: lack of quality standardization of the acquired data, technical errors in data analysis and visualization, and perhaps most importantly, the technology remains largely out of reach for common cell biology laboratories. To overcome these limitations, we developed a new experimental platform - Integrative Toolkit to Analyze Cellular Signals (iTACS). iTACS consists of two components: Acquisition and Training Module (AcTrM) and Analysis and Visualization Module (AnViM). AcTrM is based on &#181;Manager - an NIH-ImageJ-based microscope control software - and facilitates user self-training and automation of common image acquisition protocols. AnViM is based on NIH-ImageJ and facilitates user-friendly automation of data analysis and insightful visualization of results. These experiments involve culturing adherent cells on hydrogels, imaging fiducial markers embedded in the hydrogel, and finally extracting from these images a comprehensive mechanical characterization of the cells. Currently, iTACS enables the user to analyze and track a wide array of properties, including morphology, motion, cytoskeletal forces, and fluorescence of individual cells and their neighboring region. The quality standardization issue was addressed in AcTrM with, a reference image-guided refocusing technique. The technical issues in data analysis were addressed in AnViM with a multi-pronged image segmentation procedure, a user-friendly approach to identify boundary conditions, and a novel cellular property-based data visualization. AcTrM is designed to facilitate the straightforward transformation of basic fluorescence microscopes into experimental cell mechanics rigs, and AnViM is equipped to enable users to measure cellular mechanical signals without requiring an engineering background. iTACS will be available to the research community as an open-source suite with community-driven development capabilities.

The Integrative Toolkit to Analyze Cellular Signals (iTACS) platform automates the process of simultaneously measuring a wide variety of chemical and mechanical signals in adherent cells. iTACS is designed to facilitate community-driven development and enable researchers to use all platform features regardless of their educational background.

Quantitative assessment of cellular forces and motion advanced considerably over the last four decades. These advancements provided the framework to ...