This work was supported by NIH NIDCD Y1-DC-8002-01. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

1. Assembly of Probe to Stiffener

	This section of the protocol describes fabrication of a silicon stiffener, and the assembly of a thin-film polymer probe to the stiffener. Figure 1 illustrates a typical polymer neural probe along with the proposed stiffener. The details of the stiffener design are shown in Figure 2. The novel feature of this design is the shallow &quot;wicking&quot; channel running along its length which is used to distribute liquid adhesive during assembly. The wider portion of the stiffener is a tab for handling during assembly and surgical insertion. A reservoir on the tab connects to the channel. The component is fabricated from silicon using standard microfabrication processes.
<ol>
	<li>
		The silicon stiffener with a wicking channel was fabricated from a silicon-on-insulator (SOI) wafer with a device layer thickness equal to the desired thickness of the stiffener (Figure 3A). A reasonable range of stiffener thickness is 20-100 &mu;m. It is recommended that the width of the stiffener be 20-30 &mu;m smaller than the probe width, which helps to prevent overflow of the adhesive from the bond interface to the top of the probe. First the wicking channels are dry-etched using the standard Bosch process (Figure 3B). Next, the stiffener geometry is defined by a longer etch that stops on the buried oxide layer (Figure 3C). Finally, the stiffeners are released by wet-etching the buried oxide layer in 49% hydrofluoric acid (Figure 3D). After thoroughly rinsing the stiffeners, soak them in deionized water for 15 min.</li>
	<li>
		Place a pellet of polyethylene glycol (PEG) of molecular weight 10,000 g/mol into the reservoir (Figure 4). Heat the stiffener to 65 &deg;C so that the PEG melts and wicks into the channel by capillary action. Then cool to room temperature to solidify.</li>
	<li>
		Figure 5 shows a schematic of the flip chip bonder set up. Place the stiffener upside down on the base stage of the flip chip bonder, then pick up the stiffener with the tool head. Place the probe upside down on the base stage. Using the flip chip bonder, align the stiffener and the probe and then lower the stiffener and place it onto the probe.</li>
	<li>
		The base stage of the flip chip bonder should have a heating element to apply heat to the substrate. After placing the stiffener, heat the assembly once again to 65 &deg;C. Allow one minute for the PEG to remelt and begin to fill in the interface between the probe and stiffener. Cool to solidify.</li>
	<li>
		Turn the assembly over and inspect from the top. Reheat as needed to allow the PEG to completely fill the interface between the probe and the stiffener. This can be visually evaluated since the probe is transparent. As the assembly is sitting on the heater top- (probe-) side up, manually place 1-3 extra pellets of solid PEG onto the tab so that they melt over the probe, providing additional reinforcement in this region (Figure 6). Finally, allow the assembly to cool so that the PEG solidifies. At this point, the assembly is ready for surgical insertion.</li>
</ol>

	2. Insertion and Extraction
<ol>
	<li>
		Mount the probe-stiffener assembly to a micromanipulator as illustrated in Figure 7A by adhering the back of the stiffener to the micromanipulator arm at the tab region. This may be done with double-sided tape or cement, but take care not to contact the probe with adhesive. Temporarily secure the connector end of the probe to the micromanipulator with a small piece of adhesive putty such that it can be easily removed with low force.</li>
	<li>
		Position the probe assembly over the target and insert the probe with the desired insertion speed. Insertion speeds of 0.13-0.5 mm/sec were used when developing this protocol.</li>
	<li>
		Immediately remove the connector end of the probe from the micromanipulator gently and rest it on a nearby surface, such as a second manipulator arm (Figure 7B). This must be done before the PEG begins to dissolve to avoid displacing the probe.</li>
	<li>
		Allow time for PEG to dissolve. This amount of time will depend on PEG molecular weight and area of contact between the probe and stiffener. For example, with PEG molecular weight of 10,000 g/mol, a microelectrode probe about 6 mm and a matching stiffener that is 306 &mu;m wide, 15 min has been found to be an adequate amount of time. Section 3 of the protocol presents a method to test the required dissolution time. During this time, apply phosphate buffered saline (PBS) using a dropper around the tab and insertion point to dissolve any PEG that is above the target (Figure 7C).</li>
	<li>
		Using a motorized micropositioner, begin extraction of the stiffener by applying a displacement of 100 &mu;m at a speed of 5 mm/sec. This initial fast motion helps to overcome any static friction and minimize probe displacement. Then, complete the stiffener extraction at a slower speed of approximately 0.1 mm/second (Figure 7D).</li>
	<li>
		In the case of an actual surgery, continue with normal procedures to apply gel, silicone, and/or dental acrylic at the insertion site to secure and protect the probe, as demonstrated in 21.</li>
</ol>

	3. Agarose Gel Test

	This section of the protocol describes a set up and procedure to examine the extraction of the stiffener in a 0.6% agarose gel that approximates the bulk mechanical properties, pH, and salinity of brain tissue 17,22. Since the gel is nearly transparent through short distances, stiffener separation and probe displacement can be observed.
<ol>
	<li>
		Prepare a solution of 0.6% agarose in phosphate buffered saline (PBS). Mix at an elevated temperature to completely dissolve the agarose powder. Pour the solution into a shallow acrylic box; gel should be 3/4- 1 in deep. Allow to the gel set at room temperature for an hour.</li>
	<li>
		Ensure that the hardened gel is saturated with PBS so that it does not dry out, and heat the gel to 37 &deg;C.</li>
	<li>
		Set up the micromanipulator, box of agarose gel, and microscopic camera system as shown in Figure 8.</li>
	<li>
		Insert a glass reference fiducial into the box of gel by sliding it between the gel and the side of the box (Figure 8). Use a dental pick to square the features on the reference fiducial to the field of view of the digital microscope.</li>
	<li>Mount the probe assembly to the micromanipulator as described in step 2.1.</li>
	<li>Position the probe assembly over the gel about 1 mm behind the reference fiducial.</li>
	<li>Insert the probe into the gel, using the camera to guide it to a desired depth in the field of view.</li>
	<li>Immediately move the connector end of the probe to rest on a nearby surface.</li>
	<li>
		Make any required adjustments to the camera image to focus on the probe (the reference fiducial features may be slightly out of focus). Take a snapshot of the probe location.</li>
	<li>
		Allow PEG to dissolve (this time may vary, and in fact may be a parameter to be tested). Apply PBS near the tab to dissolve PEG that is above the gel.</li>
	<li>
		Start video capture if desired, and begin extraction of the stiffener as described in step 2.5. When extraction is complete, take a final snapshot of probe location.</li>
	<li>
		Use image processing tools to compare the images before and after stiffener extraction. Use the features on the reference fiducial that are visible in the field of view to register (align) the images. Calibrate the scale of the image based on the size of known features on the probe. Measure the distance of probe displacement.</li>
</ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+finite-element+model+of+the+mechanical+effects+of+implantable+microelectrodes+in+the+cerebral+cortex.">A finite-element model of the mechanical effects of implantable microelectrodes in the cerebral cortex.</a> Journal of Neural Engineering. 2, 103-113 (2005).</li><li>Lacour Sun, Y., S, , 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=Assessment+of+the+biocompatibility+of+photosensitive+polyimide+for+implantable+medical+device+use.">Assessment of the biocompatibility of photosensitive polyimide for implantable medical device use.</a> Journal of Biomedical Materials Research A. 90 (3), 648-655 (2009).</li><li>Kipke, D. R., Pellinen, D. S., Vetter, R. 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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=Ultrafast+resorbing+polymers+for+use+as+carriers+for+cortical+neural+probes.">Ultrafast resorbing polymers for use as carriers for cortical neural probes.</a> Acta Biomaterialia. 7, 2483-2491 (2011).</li><li>Gilgunn, P. J., Khilwani, R., 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=An+ultra-compliant,+scalable+neural+probe+with+molded+biodissolvable+delivery+vehicle.">An ultra-compliant, scalable neural probe with molded biodissolvable delivery vehicle.</a> , 56-59 (2012).</li><li>Ware, T., Simon, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+responsive,+softening+neural+interfaces.">Fabrication of responsive, softening neural interfaces.</a> Advanced Functional Materials. 22 (16), 3470-3479 (2012).</li><li>Harris, J. P., Capadona, J. R., 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=Mechanically+adaptive+intracortical+implants+improve+the+proximity+of+neuronal+cell+bodies.">Mechanically adaptive intracortical implants improve the proximity of neuronal cell bodies.</a> Journal of Neural Engineering. 8, 1-13 (2011).</li><li>Kozai, T. D. Y., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+shuttle+with+carboxyl+terminated+self-assembled+monolayer+coatings+for+implanting+flexible+polymer+neural+probes+in+the+brain.">Insertion shuttle with carboxyl terminated self-assembled monolayer coatings for implanting flexible polymer neural probes in the brain.</a> Journal of Neuroscience Methods. 184 (2), 199-205 (2009).</li><li>Bjugstad, K. B., Lampe, D. S., Kern, D. S., Mahoney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+of+poly(ethylene+glycol)-based+hydrogels+in+the+brain:+An+analysis+of+the+glial+response+across+space+and+time.">Biocompatibility of poly(ethylene glycol)-based hydrogels in the brain: An analysis of the glial response across space and time.</a> Journal of Biomedical Materials Research Part A. 95 (1), 79-91 (2010).</li><li>Greenwalk, R. B., Choe, Y. H., McGuire, J., Conover, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+drug+delivery+by+pegylated+drug+conjugates.">Effective drug delivery by pegylated drug conjugates.</a> Advanced Drug Delivery Reviews. 55 (2), 217-250 (2003).</li><li>Sommakia, S. S., Rickus, J. L., Otto, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+adsorbed+proteins+and+an+antifouling+agent+on+the+impedance+of+silicon-based+neural+microelectrodes.">Effects of adsorbed proteins and an antifouling agent on the impedance of silicon-based neural microelectrodes.</a> , 7139-7142 (2009).</li><li>Gage, G. J., Stoetzner, C. R., Richner, T., Brodnick, S. K., Williams, J. C., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Implantation+of+Chronic+Neural+Electrodes+for+Recording+Single+Unit+Activity+and+Electrocorticographic+Signals.">Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals.</a> J. Vis. Exp. (60), e3565 (2012).</li><li>Chen, Z. -. J., Gillies, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+realistic+brain+tissue+phantom+for+intraparenchymal+infusion+studies.">A realistic brain tissue phantom for intraparenchymal infusion studies.</a> Journal of Neurosurgery. 101 (2), 314-322 (2004).</li><li>Felix, S., Shah, K. G., George, D., Tolosa, V., Tooker, A., Sheth, H., Delima, T., Pannu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removable+silicon+insertion+stiffeners+for+neural+probes+using+polyethylene+glycol+as+a+biodissolvable+adhesive.">Removable silicon insertion stiffeners for neural probes using polyethylene glycol as a biodissolvable adhesive.</a> , 871-874 (2012).</li></ol>

The authors have no competing financial interests.

The method described here provides a well-controlled process to attach thin-film polymer probes to separate stiffeners with a biodissolvable adhesive. Also presented is the recommended surgical procedure to implement these removable stiffeners and a technique to validate the procedure in vitro for a given probe-stiffener configuration. Since the stiffener can be made arbitrarily rigid, the method can facilitate insertion of relatively long probes (&gt;3 mm). As such, the insertion method is expected to be an e...

Microelectrode arrays are an essential tool in neuroscience as well as emerging clinical applications such as prosthetics. In particular, penetrating micro-electrode probes enable stimulation and recording of neuronal activity through close contact with cells in the brain, spinal cord, and peripheral nerves. A major challenge for implanted neural probes is stability and longevity of the stimulation and recording functions. Modeling and experimental studies of the interaction between microelectrode probes and neural tissue have suggested that one mechanism for degradation is micro-tearing of neural tissue due to slight relative motion between the probe and tissue 1-3. One solution is to fabricate flexible probes that match more closely the bulk stiffness properties of neural tissue in order to minimize relative micromotion. As such, biocompatible thin film polymers such as polyimide and parylene have been adopted as favorable substrates for microelectrode probes 4-8.

	A tradeoff of flexible probes is that they are difficult to insert into the neural tissue. Researchers have taken various approaches to facilitate insertion of flexible probes while preserving the desirable mechanical properties. One class of designs modifies the polymer probe geometry to increase stiffness in certain sections or axes while maintaining compliance in other parts. This has been accomplished by incorporating ribs or layers of other materials 9,10. Another approach integrates a 3-D channel into the polymer probe design that is filled with biodegradable material 11. This probe can be temporarily stiffened, and after insertion the material in the channel dissolves and drains out. However, methods such as these that permanently modify the geometry of the final implanted device may compromise some of the desirable features of the flexible probe.

	One method that does not alter the final probe geometry is to encapsulate the polymer device with biodegradable material to temporarily stiffen the device 12-14. However, typical biodegradable materials have Young&#39;s moduli orders of magnitude smaller than that of silicon and would consequently require larger thickness to achieve the same stiffness. Adequately coating the probe can result in a more rounded or blunt tip, making insertion more difficult. Also, since dissolvable coatings are exposed, there is a risk of them dissolving immediately upon contact, or even close proximity, with the tissue.

	Another class of methods uses novel probe substrate materials that reduce in stiffness after being implanted into tissue. Such materials include shape memory polymers 15 and a mechanically adaptive nanocomposite 16. These materials are able to decrease in elastic modulus significantly after insertion, and can result in probes that more closely match the mechanical properties of neural tissue. However, the achievable range of stiffness is still limited, so they may not be able to provide very high stiffness equivalent to silicon or tungsten wires. Thus in the case of flexible probes that are very long (e.g. &gt;3 mm) or that have extremely low stiffness, a method of temporarily attaching a more rigid stiffener may still be required.

	Yet another promising method reported is to coat a stiffening shuttle with a permanent self-assembling monolayer (SAM) to customize the surface interaction between the shuttle and the flexible probe 17. When dry, the probe adheres to the coated shuttle electrostatically. After insertion, water migrates onto the hydrophilic surface, separating the probe from the shuttle so that the shuttle can be extracted. Shuttle extraction with reduced probe displacement was demonstrated (85 &mu;m). However, with only electrostatic interactions holding the probe to the shuttle, there is some risk of probe slippage relative to the shuttle before and during insertion.

	We have developed a method in which the flexible probe is attached to a stiffener with a temporary biodissolvable adhesive material that securely holds the probe during insertion. The probes used were made of polyimide, which has an elastic modulus on the order of 2-4 GPa. The stiffener was fabricated from silicon, with an elastic modulus of ~200 GPa. When attached, the stiffness of the silicon dominates, facilitating insertion. Once inserted into the tissue, the adhesive material dissolves and the stiffener is extracted to restore the probe to its initial flexibility. We selected polyethylene glycol (PEG) as the biodissolvable adhesive material. PEG has been used in implanted applications such as neural probes, tissue engineering, and drug delivery 11,18,19. Some evidence has suggested that PEG may attenuate neuroinflammatory response in brain tissue 18,20. Compared to other possible materials, including sucrose, poly lactic-co-glycolic acid (PLGA), and polyvinyl alcohol (PVA), PEG has a dissolution time in biological fluids that is of an appropriate scale for many implant surgeries (on the order of tens of minutes, depending on molecular weight). In addition, it is solid at room temperature and liquid at temperatures ranging from 50-65 &deg;C. This property makes it particularly suitable for our precision assembly process. Moreover, similar to the SAM described in 17, the dissolved PEG is hydrophilic, facilitating extraction of the stiffener. This advantageous approach is enabled by a novel stiffener design and methodical assembly process which ensure uniform adhesive coverage and accurate and repeatable alignment. In addition to the assembly process, we present the method of implementing the removable stiffener during surgery, as well as an in vitro procedure to evaluate extraction of the stiffener.

	The protocol presented herein assumes that the user possesses a flexible polymer microelectrode probe. The part of the protocol relating the fabrication of the stiffener and assembly of this probe to a stiffener assumes access to common tools found in a microfabrication facility. The protocol relating to insertion and extraction would likely be performed in a neuroscience-oriented laboratory.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Polyethylene glycol, 10,000 g/mol</td>
			<td>Sigma Aldrich</td>
			<td>309028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flexible Sub-micron Die Bonder</td>
			<td>Finetech</td>
			<td>Fineplacer lambda</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Micromanipulator</td>
			<td>KOPF</td>
			<td>1760-61</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Digital Microscope</td>
			<td>Hirox</td>
			<td>KH-7700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual Illumination Revolver Zoom Lens</td>
			<td>Hirox</td>
			<td>MXG-2500REZ</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Precision Motorized Actuator</td>
			<td>Newport</td>
			<td>LTA-HS</td>
			<td>w/ CONEX-CC controller</td>
		 </tr>
		</tbody>
 </table>

insertion of flexible neural probes using rigid stiffeners attached with biodissolvable adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

This insertion technique was used in conjunction with LLNL thin-film polyimide probes, which have passed ISO 10993 biocompatibility standards and are intended for chronic implantation. A typical thin-film polyimide probe is illustrated in Figure 1 along with a silicon stiffener that is approximately 10 mm long in the narrow region. This stiffener has one wicking channel running along its length, as shown in Figure 2. Figure 3 illustrates the mimcrofabrication process u...

Watch this Scientific Journal Video about Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive at JoVE.com

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

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14.6K Views.  Lawrence Livermore National Laboratory. Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Video: Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing the complicated multi-bracket appliances to a simple two-bracket system for the purpose of force system evaluation will be the first step in this direction. However, much of the orthodontic biomechanics in this regard is confined to 2D experimental studies, computer modeling/analysis or theoretical extrapolation of existing models. The objective of this protocol is to design, construct and validate an in vitro 3D model capable of measuring the forces and moments generated by an archwire with a V-bend placed between two brackets. Additional objectives are to compare the force system generated by different types of archwires among themselves and to previous models. For this purpose, a 2 x 4 appliance representing a molar and an incisor has been simulated. An orthodontic wire tester (OWT) is constructed consisting of two multi-axis force transducers or load cells (nanosensors) to which the orthodontic brackets are attached. The load cells are capable of measuring the force system in all the three planes of space. Two types of archwires, stainless-steel and beta-titanium of three different sizes (0.016 x 0.022 inch, 0.017 x 0.025 inch and 0.019 x 0.025 inch), are tested. Each wire receives a single vertical V-bend systematically placed at a specific position with a predefined angle. Similar V-bends are replicated on different archwires at 11 different locations between the molar and incisor attachments. This is the first time an attempt has been made in vitro to simulate an orthodontic appliance utilizing V-bends on different archwires.

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...