Alberto Guiggiani for developing the real time control system, Evelina Chieregatti and Hanako Tsushima for insightful discussions, Giacomo Pruzzo and Alessandro Parodi for the development of custom electronics and software, and Claudia Chiabrera and Marina Nanni for their expert advice and assistance in cell culture preparation.

1. Optical Setup
The entire optical system was described earlier15. Briefly, the optical tweezers system is based on an ytterbium continuous wave (CW) fiber laser operating at 1064 nm (IPG Laser GmbH). A spatial light modulator (SLM) (LCOS-SLM, model X10468-07 - Hamamatsu) varies the phase of the incoming IR laser beam to control the position of the trapping focus spot on the culture dish by computer generated holograms. The freely available Blue-tweezers software (web link on equipment table) generated holograms projected on the spatial light modulator. The interferometer for force spectroscopy measurements was based on a four-quadrant photodiode (QPD, S5980 with C5460SPL 6041 board - Hamamatsu) and a photodiode (PD, PDA100A-EC - Thorlabs).
The laser dissection source was a pulsed sub-nanosecond UV Nd:YAG laser at 355 nm (PNV-001525-040, PowerChip nano-Pulse UV laser - Teem Photonics). An acousto-optic modulator (MQ110-A3-UV, 355 nm fused silica -AA-Opto-electronic) controlled the power of the UV laser delivered to the sample.
The holographic optical tweezers and laser micro-dissector were integrated on a modified upright microscope (BX51 - Olympus) equipped with a 60X, 0.9 NA water dipping objective.The stage of the microscope is composed of a 3-axis linear DC motor micro-positioning system (M-126.CG1, Physics-Instruments) carrying a separate 3-axis piezoelectric nano-positioning stage (P-733.3DD, Physics-Instruments) to combine coarse movement of the sample with the sub-nanometer resolution of the piezo-stage. The microscope stage system was equipped with two control loops synergistically acting to maintain the trapping focus spot at the right position, depending on the selected working mode (position or force clamp, static or dynamic)16. In particular, an internal feedback loop acts on a piezoelectric stage, to keep the bead at a selected distance from the trap center. The other external loop controls the position of the motorized stage to exploit the region spanned by the piezo-actuator on a larger area than its available stroke17. When the piezo-stage reaches the limit of the available stroke in one direction, the external loop moves the micro stage in the opposite direction, thus the piezo recovers toward its central position because it is tracking the trapped bead adhered to the sample. When the piezo-stage reaches the central position of its course range, the micro stage halted. Further details of the system are reported in Guiggiani et al16,17.
A Peltier device (QE1 resistive heating with TC-344B dual channel heater controller - Warner Instruments) controls the temperature of the cell culture under the microscope (37 &deg;C). In the culture, pH and humidity were maintained at physiological conditions by aerating a custom-designed polydimethylsiloxane (PDMS) sleeve (integrating the microscope objective) with humidified carbogen (95% O2, 5% CO2).
2. Cell Culture Preparation
All the experimental protocols were approved by the Italian Ministry of Health. Primary cultures were obtained from hippocampi of mice (C57BL6J, Charles River) at embryonic day 18 (E18).
<ol>
	<li>Neurons were plated at a concentration of 25,000 cells/ml on glass-bottom Petri dishes (P35G-0-14-C - MaTek Corporation). The low concentration of cultured cells is needed to avoid the formation of a dense network already in the first days in vitro. At the mentioned cell concentration, the likelihood of finding isolated cells with a longer neurite not connected to other cells is much higher.</li>
</ol>
3. Bead Coating
<ol>
	<li>Polymer microspheres (&Oslash; 4 &mu;m, COOH-terminated- Bangs Laboratories, product code PC05N/6700) were coated with poly-D-lysine following the procedure described in the polyLink Protein Coupling Kit (Polysciences, product code 19539). The beads are coated with the same molecule used to cover the culture support and favor cells adhesion.</li>
</ol>
4. Choose Isolated Neuron. Detach A Bead from the Culture Substrate, Trap and Move it Next to the Neuron
<ol>
	<li>Put the culture Petri dish on the microscope stage. After setting the focus on the sample by stage motion, correct the position of the illuminating objective condenser to set Kohler-illumination. Aligning the illumination optics to set the Kolher-illumination condition is necessary to improve the bright field imaging quality. Using such alignment conditions, it is also necessary to maximize the IR laser collection efficiency (through objective condenser), from the scattering trapped probe, to perform its interferometric tracking by the QPD and PD.</li>
	<li>Pipette a few &mu;l of the coated microsphere stock solution into the culture dish. Microspheres will deposit and adhere to the culture substrate. The number of &mu;l injected into the culture dish depends on the microsphere concentration of the stock solution. The ideal condition is to have between one and two microspheres per imaging field of view. When too many microspheres are added to the culture dish, they can already attach randomly to the cultured neurons.</li>
	<li>Move around in the culture dish, searching for an isolated neuron with a longer neurite (the axon), and save the position of the microscope stage.</li>
	<li>Move around and search for a bead adhered to the culture support.</li>
	<li>Turn on the IR trapping laser. At this stage, no holograms are projected on SLM, and the position of IR laser spot coincides with the UV laser spot position. Set the axial position of the IR spot 2-3 &mu;m above the culture support surface, by microscope stage motion.</li>
	<li>Set the UV laser power delivered to the sample to less than 1 &mu;W. The UV laser dissector has been previously calibrated14: 
	5-6 &mu;W the glass support is ablated 
	4 &mu;W a neuronal connection is completely dissected 
	2.5 &mu;W a neurite is partially lesioned 
	&lsaquo; 1 &mu;W a shock wave is produced in the culture dish. The optical shock wave is strong enough to detach the bead from the glass support.</li>
	<li>Turn on the UV laser, while leaving the IR laser on, and move the UV spot above the adhered bead by microscope stage motion. The bead detaches from the surface, and it is trapped by the IR laser. Then, turn off the UV laser.</li>
	<li>Move the trapped bead several micrometers above the glass support (20-30 &mu;m). Lifting the bead to this position will prevent it from contacting other cells while it is being moved toward the previously chosen neuron. Bring the bead to the previously saved stage position. Set the stage speed to a low value (10 &mu;m/sec) so the drag fluid force does not exceed the optical trapping force.</li>
</ol>
5. Move the Trap Position with Respect to the Laser Dissector Spot and Axon Position by Computer Generated Hologram, and Calibrate the Optical Tweezers Stiffness
<ol>
	<li>Move the bead toward the glass support to visualize the axon. The bead is held above the cell to avoid contact with it (about 5 &mu;m above the glass support).</li>
	<li>Move the microscope stage to move the UV focus spot on the center of the width of the axon. Save the current stage position.</li>
	<li>Move the IR focus spot position by computer-generated hologram. In this step, the stage is halted in the position chosen in the previous step. When the computer generated hologram is projected on the SLM, the trapped bead is moved with respect to the axon. We move the IR laser spot to have the trapped bead aligned on the center of the neurite, with the UV laser spot centered on the same neurite too. The IR spot and thus the trapped bead are positioned along the neurite 5-10 &mu;m away from the UV spot. We move the position of the IR with respect to the UV spot depending on the neurite geometry. In case the optical tweezers are not equipped with an SLM system, the position can be varied by tilting mirrors, or acoustic optic deflectors, on the IR optical path. The IR laser spot may be positioned 5-10 &mu;m away from the UV spot to avoid optical interaction of the dissecting beam with the trapped probe, which could influence its Brownian motion and alter the force spectroscopy traces.</li>
	<li>After defining the new IR spot position with respect to UV spot and axon position, move the stage to position the trapped bead away from the axon and avoid collision with it. Move the axial position of the trapped bead to about 2 &mu;m above the cover glass.</li>
	<li>Align the QPD to center the interference fringes on it: x and y QPD differential signals are zeros when the QPD is centered. Acquire 5 sec of the Brownian motion of the trapped bead by the interferometer, at 50 KHz sampling rate. Obtain the stiffness (pN/nm) and sensitivity (V/nm) of the optical trap by the power spectrum method18.</li>
</ol>
6. Attach the Bead to the Axon. Perform Axotomy and Simultaneous Force Measurement
<ol>
	<li>Raise the trapped bead position about 4 &mu;m above the cover glass, and move it to the previously saved stage position (see step 3.2).</li>
	<li>Move the trapped bead down toward the axon until it contacts the neurite. The collision between the trapped bead and the axon is monitored by the z QPD signal detecting displacement of the trapped bead in the axial direction.</li>
	<li>Wait 10 sec with the trapped bead pushed against the axon to favor its adhesion to the neurite membrane.</li>
	<li>Move the micro stage to displace the trapped bead from the axon, and thus verify its adhesion to the cell membrane. If the bead adhered, it escapes from the optical trap.</li>
	<li>Move back the laser trap on the adhered bead and switch on the force-clamp loop with force condition equal to zero. In such a way, the piezo-stage repositions the bead, attached to the cell, on the optical trap center. If the system does not have a feedback loop control, the laser trap position can be moved by the microscope stage to center it on the adhered probe. The center of the trap is reached when the QPD signals are the same as when the bead is trapped and not adhered to the cell (x and y QPD signals equal to zero, and the z QPD signal gives the same voltage sum of the four quadrants as when the bead is trapped far from any surface) .</li>
	<li>Set a force-clamp condition on the z axis, positioning the trapping laser slightly over the center of the bead (about 100 nm), to generate a pretension on the adhered trapped probe. In case the system is not equipped with a force-clamp control, the optical trap position can be raised up at steps of 25 nm by the microscope stage. The z QPD signal allows monitoring. Therefore, use this signal to set the position of the laser trap with respect to the adhered probe. Such pre-tension is needed to sense the strain on the membrane after the axonal lesion, and the consequent decrease of Brownian motion of the adhered probe. A similar approach has been proposed to measuring the release of tension in a membrane tether by video imaging19.</li>
	<li>Switch-off the force-clamp feedback to measure the force on the adhered probe in the position-clamp condition (the piezo-stage is blocked).</li>
	<li>Start simultaneous recording of the trapped probe positions through the interferometer (20 KHz sampling frequency), and the cell during laser axotomy time-lapse bright-field imaging (20 Hz frame rate).</li>
	<li>Turn on the UV laser to deliver laser pulses until a lesion becomes visible on the image of the axon (Usually 200-400 optical pulses are needed. Energy per pulse: 25 nJ), then turn off the UV laser.</li>
	<li>Continue recording by the interferometer for about 3 min, until the x, y and z QPD traces reach a plateau.</li>
</ol>
7. Quantify the Total Tension Release
<ol>
	<li>Convert the recorded QPD traces (in Volts) by the calibrated sensitivity of the optical trap, to obtain the respective traces in nanometers.</li>
	<li>Filter the x, y, and z displacement traces by a high pass filter with cut off at 10 Hz.</li>
	<li>Calculate the total variance of the filtered traces representing the Brownian motion of the trapped bead. Calculate the variance at 25 msec steps for overlapping time windows of 500 msec (10,000 data points)20. The high pass filtering of traces excludes the changes of Brownian motion due to the membrane fluctuation or cortical actin motion. The Brownian motion of the bead is reduced by the optical forces and the adhesion forces on the cell membrane. When the membrane is strained because of the release of tension its viscosity is increased, and thus the Brownian motion of the bead, detected immediately after axotomy, starts to decrease.</li>
	<li>Define t0: the beginning of decrease of the Brownian motion variance, after the delivery of UV laser energy. The time instant t0 indicates the beginning of membrane strain.</li>
	<li>Define t1: the end of decrease of the Brownian motion variance (when it reaches a plateau). The time instant t1 indicates the end of membrane strain.</li>
	<li>Perform video tracking of debris or a scratch on the cover glass support, to measure any drift of the sample during the force measurement. To track the particle on the glass support, we first apply thresholds to the bright-field images, to obtain a stack of binary images with the particle in white on a black background. Then, we track the particle center of mass with sub-pixel accuracy (using the freeware ImageJ software).</li>
	<li>Subtract the measured drift from the displacement QPD traces. Multiply the drift-corrected QPD traces by the respective calibrated optical stiffness (kx, ky, kz) to obtain the Fx, Fy, Fz traces in piconewtons.</li>
	<li>Calculate the total force trace as Ftot = &radic;(Fx2+Fy2+Fz2).</li>
	<li>Calculate the total release of force between t0 and t1 as Freleased= Ftot(t1) - Ftot(t0). The force measured at t0 has to be subtracted because it is due to the displacement of the probe from the trap center, when the bead adheres to the neurite.</li>
	<li>Calculate the neurite contact area between the trapped bead and the UV laser spot position: on the bright field image measure the long (L) and short (D) axes of the neurite between the trapped bead and the UV laser spot position. The axon contact area is Aaxon = L x D.</li>
	<li>Normalize the total released force Freleased by the axon contact area Aaxon.</li>
</ol>

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The authors declare that they have no competing financial interests.

We report in this work a quantitative method to compare the neurite adhesion to the culture substrate, by performing simultaneous force spectroscopy measurement during laser induced cell lesion. The measured release of tension is related to the degree of adhesion of the cell to the substrate: cells with a higher number of focal adhesions should release less tension. Measuring the release of tension in terms of piconewtons provides a physical quantity by which to evaluate the axonal adhesion to the culture support in diff...

Optical microscopy is one of the less invasive imaging system available to observe living cells. With the exploitation of effects such as radiation pressure (as in optical tweezers1), or high-energy photon flux (as in laser dissector2), this technology was extended to nano-manipulation. The optical imaging system furnishes a precise control to visualize and manipulate sub cellular targets3. At the same time, thanks to the accurate calibration of the delivered laser power, optical tools accomplish either soft or invasive sample manipulation with unprecedented reproducibility.
Several laboratories integrated, in the same experimental setup, optical tweezers and laser dissector in order to ablate organelles4, to fuse together different cells5, or to stimulate cells by optically driven cargos6,7. While optical tweezers, after calibration of the optical stiffness, allow for the control of applied force to the cell on a piconewton scale, laser dissection systems can modulate optical manipulation, which ranges from membrane photo-poration to ablation of single organelles or dissection of sub-cellular structures. However, laser dissection calibration relies on qualitative assessment of the entity of optical manipulation with respect to the energy delivered to the sample, mainly based on image analysis illustrating morphological changes caused to the specimen8. In the presented method, we demonstrate how to perform force spectroscopy measurement during the laser axonal dissection of a developing neuron, to quantify, on piconewton scale, the force produced by an altered equilibrium in the cytoskeleton structure of a sub-cellular compartment9. Cultured neurons adhere to the substrate, and polarize during development. The polarization phase occurs during the first five days in vitro. At stage two of polarization, one of the extruding neurites becomes longer, and it will differentiate to become the axon10. Axonal elongation in response to towing force at the growth cone has been previously modeled by Dennerl's model11. Recently, this model has been extended12 to include the role of neurite adhesion to the extracellular matrix substrates. This biophysical model, proposed after experimental observations13, showed that pulling forces on growth cone, propagating along the neurite, are modulated by focal adhesions to the substrate. Likewise, axonal lesion produces a local release of tension propagating toward the cell body. Thus, we proposed that measuring such released tension in a location along the axon between the lesion and the cell soma offers the possibility to assess the dampening outcome of unaffected focal adhesions.
We calibrate the necessary energy photon-flux of the laser dissector to control the extent of the inflicted axonal damage, from complete transection to partial lesion. Following the calibration, we repeated partial lesion to the axons of several differentiating neurons and developed the protocol to quantify the tension release, and thus obtained a quantitative parameter to estimate the adhesion of the axon to the substrate14.
In the present work, we describe in detail the developed protocol, which represents a precise experimental procedure to evaluate and compare with piconewton sensitivity the axonal adhesion to the substrate in different experimental conditions such as chemical treatment14, or different types of cell culture support.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Polymer microspheres, &Oslash; 4 &mu;m, COOH coated</td>
			<td>Bangs laboratories</td>
			<td>PC05N/6700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PolyLink Protein Coupling Kit</td>
			<td>Polyscience</td>
			<td>19539</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>IR laser</td>
			<td>IPG Laser GmbH</td>
			<td>YLM-5-SC-LP</td>
			<td>ytterbium continuous wave (CW) fiber laser operating at 1064 nm, with linear polarization</td>
		</tr>
		<tr>
			<td>Spatial light modulator</td>
			<td>Hamamatsu</td>
			<td>LCOS-SLM 10468-07</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue-tweezers software</td>
			<td>Optics group, University of Glasgow</td>
			<td>Free downloadable software</td>
			<td><a href="http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/" target="_blank">http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/</a></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>Free downloadable software</td>
			<td><a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/ </a></td>
		</tr>
		<tr>
			<td>QPD</td>
			<td>Thorlabs</td>
			<td>S5980 with C5460SPL 6041 board</td>
			<td>Four quadrant photo-diode to measure x, y trapped probe displacement</td>
		</tr>
		<tr>
			<td>PD</td>
			<td>Teem Photonics</td>
			<td>PDA100A-EC</td>
			<td>Photodiode to measure z trapped probe displacement</td>
		</tr>
		<tr>
			<td>nano-Pulse UV laser</td>
			<td>AA-optoelctronics</td>
			<td>PNV-001525-040</td>
			<td>Pulsed UVA laser, pulse length 400 ps</td>
		</tr>
		<tr>
			<td>Acoustic Optic Modulator</td>
			<td>Olympus</td>
			<td>MQ110-A3-UV, 355 nm fused silica</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Andor</td>
			<td>BX51</td>
			<td>Equipped with a 60X, 0.9 NA, water dipping objective</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>Warner Instruments</td>
			<td>V887ECSUVB EMCCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peltier device</td>
			<td>Physic Instruments</td>
			<td>QE1 resistive heating with TC-344B dual channel heater controller</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope stage: micro+piezo stage</td>
			<td>National Instruments</td>
			<td>Three linear stages M-126.CG1 carrying a separate 3-axis piezoelectric nano-positioning stage P-733.3DD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Daq</td>
			<td>&nbsp;</td>
			<td>NI PCI-6229</td>
			<td>Acquiring the x, y, z position of the trapped probe, and sending feedback loop signals to microscope stage</td>
		</tr>
		<tr>
			<td>Linux Real Time Application Interface (RTAI) machine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Real time feedback loop system, to control stage position, developed on a dedicated PC desktop</td>
		</tr>
	</tbody>
</table>

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.

The cell generates traction forces on the substrate by its focal adhesions. Force generated by cytoskeletal elements are in equilibrium with the counteracting force of the culture substrate. After laser induced lesion of the neurite, some of the tensed cytoskeleton cables are disrupted and their equilibrated tension is released because the opposing force of the substrate adhesion is eliminated. The released tension is partially distributed on the unaffected focal adhesions, and the bead attached to the cell membrane, hel...

Watch this Scientific Journal Video about Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution at JoVE.

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 substrate.

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

measurement-tension-release-during-laser-induced-axon-lesion-to

Research

JoVE Journal

Bioengineering

10.2K 조회수.  National Research Council of Italy. 우리는 부분적으로 축삭의 세포막에 부착 광학적 포획 프로브에서 수행 동시 힘 분광 측정으로 레이저 해부학자로 lesioned 된 축삭의 긴장 방출을 측정 하였다. 개발 된 실험 프로토콜은 문화 기판에 축삭 접착력을 평가합니다.

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

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.

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 ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

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

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

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

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