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>&#8206;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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7.5K 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

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

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

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

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

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

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

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

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

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

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

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

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

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

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

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

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...