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: ...

<ol>
	<li>Price, L. S., Leng, J., Schwartz, M. A., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Rac+and+Cdc42+by+Integrins+Mediates+Cell+Spreading.">Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading.</a> Molecular Biology of the Cell. 9 (7), 1863-1871 (1998).</li><li>Cavalcanti-Adam, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Spreading+and+Focal+Adhesion+Dynamics+Are+Regulated+by+Spacing+of+Integrin+Ligands.">Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands.</a> Biophysical Journal. 92 (8), 2964-2974 (2007).</li><li>Huttenlocher, A., Horwitz, A. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA-based+molecular+probe+for+optically+reporting+cellular+traction+forces.">A DNA-based molecular probe for optically reporting cellular traction forces.</a> Nature Methods. 11 (12), 1229-1232 (2014).</li><li>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=Integrins+outside+focal+adhesions+transmit+tensions+during+stable+cell+adhesion.">Integrins outside focal adhesions transmit tensions during stable cell adhesion.</a> Scientific Reports. 6 (1), 36959 (2016).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-activatable+biosensor+enables+single+platelet+force+mapping+directly+by+fluorescence+imaging.">Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.</a> Biosensors and Bioelectronics. 100, 192-200 (2018).</li><li>Zhao, Y., Wang, Y., Sarkar, A., 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=Keratocytes+Generate+High+Integrin+Tension+at+the+Trailing+Edge+to+Mediate+Rear+De-adhesion+during+Rapid+Cell+Migration.">Keratocytes Generate High Integrin Tension at the Trailing Edge to Mediate Rear De-adhesion during Rapid Cell Migration.</a> iScience. 9, 502-512 (2018).</li><li>Crisalli, P., Kool, E. 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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, ...

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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 ...

This DNA-based integrative tension sensor, or ITS, converting force signal to fluorescence locally enables the direct imaging of cellular force with fluorescence microscopy. ITS has a high spatial resolution and a high sensitivity. The activation threshold is tunable, allowing selective image of integrin tension at different levels.After receiving the customized single-stranded DNA, or ssDNA, of interest, to conjugate RGD peptide to the thiolated ssDNA quencher, first add 100 microliters of 0.5-molar EDTA in 400 microliters of water to about 450 microliters of freshly prepared TCEP solution. Then, add 10 microliters of the TCEP-EDTA solution to 20 microliters of one-millimolar thiol DNA quencher in PBS for a 30-minute incubation at room temperature. To conjugate RGD amine with sulfo-SMCC, add 40 microliters of freshly prepared 23-millimolar sulfo-SMCC to 100 microliters of freshly prepared 11-millimolar RGD amine solution for a 20-minute incubation at room temperature.To conjugate RGD amine SMCC with the thiolated ssDNA quencher, mix the entire volume of the thiolated ssDNA quencher solution with the entire volume of the RGD amine SMCC solution for a one-hour incubation at room temperature. At the end of the incubation, transfer the solution to four-degree Celsius storage overnight. The next morning, mix three-molar sodium chloride with the thiol-conjugated DNA solution and minus 20-degree Celsius, chilled, 100%ethanol at a one-to-10-to-25 ratio, and place the reaction at minus 20 degrees Celsius for about 30 minutes.When the DNA has precipitated, collect the DNA by centrifugation, and resuspend the pellet in PBS for measurement of the DNA concentration on a spectrometer. For integrative tension sensor, or ITS, synthesis, mix the upper and lower strand DNA solutions at a 1.1-to-one-molar ratio, and incubate the reaction overnight at four degrees Celsius before placing aliquots of the mixture at minus 20 degrees Celsius for long-term storage. For ITS immobilization, coat a 29-millimeter-diameter, glass-bottom Petri dish with 200 microliters of 0.1-milligram-per-milliliter biotinylated bovine serum albumin, or BSA-biotin, for 30 minutes at four degrees Celsius.When the BSA-biotin has physically adsorbed on the glass surface, wash the dish three times with 200 microliters of fresh PBS per wash, followed by incubation with 200 microliters of 50 micrograms per milliliter of avidin protein for 30 minutes at four degrees Celsius. At the end of the incubation, wash the dish three times with PBS as demonstrated, and add 200 microliters of 0.1-micromolar ITS for another 30-minute incubation at four degrees Celsius. At the end of the incubation, wash the dish three times with PBS, leaving the last wash in the dish until the cell plating.To initiate a fish keratocyte culture, use a flat-tipped tweezer to pluck a piece of scale from a fish, and gently press the scale onto the well of an unmodified glass-bottom Petri dish, with the inner side of the scale contacting the glass. Wait for about 30 seconds for the fish scale to adhere to the glass surface of the dish before covering the scale with one millimeter of complete Iscove's Modified Dulbecco's Medium. Then, incubate the scale in a dark and humid box for six to 48 hours at room temperature.To plate the keratocytes onto the ITS surface, replace the medium from the keratocyte culture dish with 1X cell detaching solution for a three-minute incubation at room temperature. At the end of the incubation, replace the detaching solution with fresh complete medium, and dissociate the cells with gentle pipetting. Then, adjust the cells to the appropriate concentration for plating, and seed them onto the ITS coated dish for 30 minutes at room temperature before imaging.For quasi-real-time integrin tension imaging in live cells, transfer the cells to the stage of a fluorescence microscope, and select the 40 or 100 times objective. In the imaging software, set the exposure time to one second and the frame interval of 20 seconds to obtain a video of the ITS images. Then, use an appropriate image analysis software program 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 quasi-real-time manner. Here, integrin tension mapping demonstrates the induction of integrin tension at two force tracks during keratocyte migration. The resolution of the force map could then be calibrated to be 0.4 micrometers.High integrin tension concentrates at the rear margin in motile fish keratocytes. In nonmotile cells, a specific integrin tension pattern that is quite different from that observed in the fast migrating keratocyte is formed. Focal adhesions and the integrin tension of fish keratocytes can be co-imaged to evaluate the relationship between integrin tension and cell structure in cells of interest.With ITS, cell adhesion force becomes visible with a resolution and a sensitivity comparable to cell structural imaging. This technique paves the road for the study of a force-structure interplay in cells.

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7.5K ビュー.  Iowa State University. このDNAベースの統合張力センサ(ITS)は、力信号を蛍光に局所的に変換することで、蛍光顕微鏡による細胞力の直接イメージングを可能にする。ITSは高い空間分解能と高感度を有する。活性化閾値は調整可能であり、異なるレベルでのインテグリン張力の選択的なイメージを可能にする。カスタマイズされた一本鎖DNA(ssDNA)を受け取って、RGDペプチドをチオラ化ssDNAク?...

ビデオ: 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...

an in vitro assay to study platelet migration using rgd-functionalized avidin-biotin tethers

Author Spotlight: Role of Integrin Ligand Density in Platelet Migration and Force Generation on Inflamed Blood Vessels

A detailed protocol for imaging single migrating platelets using RGD-functionalized avidin-biotin tethers with tunable density is provided, revealing that platelets generate enough force to rupture the avidin-biotin...

Imaging Single Platelet Migration Using a Three-Layer Coating System

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

tension gauge tether probes for quantifying growth factor mediated integrin mechanics and adhesion

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell...

measurement of tension release during laser induced axon lesion to evaluate axonal adhesion to the substrate at piconewton and millisecond resolution

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

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...

in vivo imaging of intact drosophila larvae at sub-cellular resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures  for up to a few...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion...

sub-nanometer resolution imaging with amplitude-modulation atomic force microscopy in liquid

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution...

ultrasonic fatigue testing in the tension-compression mode

Ultrasonic Fatigue Testing in the Tension-Compression Mode

A protocol for ultrasonic fatigue testing in the high and ultra-high cycle region in axial tension-compression loading...

proprioception and tension receptors in crab limbs: student laboratory exercises

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking...

integrative toolkit to analyze cellular signals: forces, motion, morphology, and fluorescence

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

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...

generation of induced pluripotent stem cell-derived itenocytes via combined scleraxis overexpression and 2d uniaxial tension

Author Spotlight: Advancements in Cell and Tissue Engineering for Tendon Repair 

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D...

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

accurate determination of the equilibrium surface tension values with area perturbation tests

Accurate Determination of the Equilibrium Surface Tension Values with Area Perturbation Tests

Two protocols for determining the equilibrium surface tension (EST) values using the emerging bubble method (EBM) and the spinning bubble method (SBM) are presented for a surfactant-containing aqueous phase against...

dna tension probes to map the transient piconewton receptor forces by immune cells

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

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

imaging cleared embryonic and postnatal hearts at single-cell resolution

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

peering at brain polysomes with atomic force microscopy

Peering at Brain Polysomes with Atomic Force Microscopy

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

optogenetic inhibition of rho1-mediated actomyosin contractility coupled with measurement of epithelial tension in drosophila embryos

Author Spotlight: Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos  

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in...

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

micromechanical tension testing of additively manufactured 17-4 ph stainless steel specimens

Micromechanical Tension Testing of Additively Manufactured 17-4 PH Stainless Steel Specimens

Presented here is a procedure for measuring fundamental material properties through micromechanical tension testing. Described are the methods for micro-tensile specimen fabrication (allowing rapid micro-specimen fabrication from bulk material volumes by combining photolithography, chemical etching, and focused ion beam milling), indenter tip modification, and micromechanical tension testing (including an...

sample preparation for single virion atomic force microscopy and super-resolution fluorescence imaging

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence...

tension-free weight-bearing model of steroid-induced osteonecrosis of femoral head in rats

Author Spotlight: Developing a Rat Model for Weight-Bearing Intervention to Investigate Osteonecrosis of the Femoral Head

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic...

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

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 ...

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 ...