Name: micro-particle image velocimetry for velocity profile measurements of micro blood flows
Uploaded: 2013-04-25T12:00:00
Description: Watch this Scientific Journal Video about Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows at JoVE.com

The authors would like to thank NSERC (Natural Sciences and Engineering Council of Canada) for funding, Catherine Pagiatikis for her help in initial runs, Sura Abu-Mallouh and Frederick Fahim for testing the protocol, Richard Prevost of LaVision, Inc for technical support, and Guy Cloutier of the University of Montr&eacute;al for the loan of the Dalsa high-speed camera.

1. Microchip Fabrication
The first step is to create or purchase your microchannel. There are many options for microchip material.
One of the most common materials chosen is poly(dimethylsiloxane) (PDMS). There are many publications on directions for PDMS fabrication through soft lithography 16,17,18.
Once the PDMS channel is fabricated, there are several surface treatments available to reverse its natural hydrophobicity. Oxygena...

<ol>
	<li>Santiago, J. G., Wereley, S. T., Meinhart, 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=A+particle+image+velocimetry+system+for+microfluidics.">A particle image velocimetry system for microfluidics.</a> Experiments in Fluids. 25, 316-319 (1998).</li><li>Sugii, Y., Okamoto, K., Nishio, S., Nakano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Velocity+Measurement+in+Micro+Tube+by+Highly+Accurate+PIV+Technique.">Evaluation of Velocity Measurement in Micro Tube by Highly Accurate PIV Technique.</a> , 1-5 (2001).</li><li>Park, J. S., Choi, C. K., Kihm, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+sliced+micro-PIV+using+confocal+laser+scanning+microscopy+(CLSM).">Optically sliced micro-PIV using confocal laser scanning microscopy (CLSM).</a> Exp. Fluids. 37 (1), 105-119 (2004).</li><li>King, M. R., Bansal, D., Kim, M. B., Sarelius, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hematocrit+and+leukocyte+adherence+on+flow+direction+in+the+microcirculation.">The effect of hematocrit and leukocyte adherence on flow direction in the microcirculation.</a> Ann. of Biomed. Eng. 32 (6), 803-814 (2004).</li><li>Lima, R., Wada, S., Tsubota, K., Yamaguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+micro-PIV+measurements+of+three-dimensional+profiles+of+cell+suspension+flow+in+a+square+microchannel.">Confocal micro-PIV measurements of three-dimensional profiles of cell suspension flow in a square microchannel.</a> Meas. Sci. Tech. 17 (4), 797-808 (2006).</li><li>Wereley, S. T., Santiago, J. G., Chiu, R., Meinhart, C. D., Adrian, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-resolution+particle+image+velocimetry.">Micro-resolution particle image velocimetry.</a> Micro- and Nanofabricated Structures and Devices for Biomedical Environmental Applications. 3258, 122-133 (1998).</li><li>Meinhart, C. D., Wereley, S. T., Gray, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+illumination+for+two-dimensional+particle+image+velocimetry.">Volume illumination for two-dimensional particle image velocimetry.</a> Measurement Science and Technology. 11, 809-814 (2000).</li><li>Chayer, B., Pitts, K. L., Cloutier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Velocity+measurement+accuracy+in+optical+microhemodynamics:+experiment+and+simulation.">Velocity measurement accuracy in optical microhemodynamics: experiment and simulation.</a> Physiological Measurement. , (2012).</li><li>Tabeling, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Microfluidics. , (2005).</li><li>Olson, M. G., Adrian, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out-of-focus+effects+on+particle+image+visibility+and+correlation+in+microscopic+particle+image+velocimetry.">Out-of-focus effects on particle image visibility and correlation in microscopic particle image velocimetry.</a> Experiments in Fluids. 29, S166-S174 (2000).</li><li>Wereley, S. T., Meinhart, 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=Recent+advances+in+micro-particle+image+velocimetry+techniques.">Recent advances in micro-particle image velocimetry techniques.</a> Ann. Rev. Fluid Mech. 42, 557-576 (2010).</li><li>Williams, S. J., Park, C., Wereley, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+applications+on+microfluidic+velocimetry.">Advances and applications on microfluidic velocimetry.</a> Microfluid. Nanofluid. 8 (6), 709-726 (2010).</li><li>Chiu, J. J., Chen, S. <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+disturbed+flow+on+vascular+endothelium:+Pathophysiological+basis+and+clinical+perspectives.">Effects of disturbed flow on vascular endothelium: Pathophysiological basis and clinical perspectives.</a> Physiol. Rev. 91 (1), 327-387 (2011).</li><li>Secomb, T. W., Pries, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microcirculation:+Physiology+at+the+mesoscale.">The microcirculation: Physiology at the mesoscale.</a> J. Physiol. 589 (5), 1047-1052 (2011).</li><li>Popel, A. S., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculation+and+hemorheology.">Microcirculation and hemorheology.</a> Annual Review of Fluid Mechancis. 37, 43-69 (2005).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography.">Soft Lithography.</a> Angewandte Chemie International Edition England. 37, 551-577 (1998).</li><li>Whitesides, G. M., Stroock, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+methods+for+microfluidics.">Flexible methods for microfluidics.</a> Physics Today. , 42-48 (2001).</li><li>Sia, S. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+of+poly(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated of poly(dimethylsiloxane) for biological studies.</a> Electrophoresis. 24, 3563-3576 (2003).</li><li>Zhou, J., Ellis, A. V., Voelcker, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+PDMS+surface+modifications+for+microfluidic+devices.">Recent developments in PDMS surface modifications for microfluidic devices.</a> Electrophoresis. 31, 2-16 (2009).</li><li>Pitts, K. L., Abu-Mallouh, S., Fenech, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+angle+study+of+blood+dilutions+on+common+microchip+materials.">Contact angle study of blood dilutions on common microchip materials.</a> Journal of the Mechanical Behavior of Biomedical Materials. , (2012).</li><li>Hovav, T., Yedgar, S., Manny, N., Barshtein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+red+blood+cell+aggregability+and+shape+during+blood+storage.">Alteration of red blood cell aggregability and shape during blood storage.</a> Transfusion. 39, 277-281 (1999).</li><li>Pitts, K. L., Mehri, R., Mavriplis, C., Fenech, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-particle+image+velocimetry+measurement+of+blood+flow:+validation+and+analysis+of+data+pre-processing+and+processing+methods.">Micro-particle image velocimetry measurement of blood flow: validation and analysis of data pre-processing and processing methods.</a> Measurement Science and Technology. 23, 105302 (2012).</li><li>Kloosterman, A., Poelma, C., Westerweel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+rate+estimation+in+large+depth-of-field+micro-PIV.">Flow rate estimation in large depth-of-field micro-PIV.</a> Exp. Fluids. 50 (6), 1587-1599 (2011).</li><li>Goldsmith, H. L., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics.">Hemodynamics.</a> Annual Review of Fluid Mechanics. 7, 213-247 (1975).</li></ol>

Using &mu;PIV for blood flow measurements at the scale of the microcirculation can give insight into a great number of relevant biomedical, mechanical and chemical engineering processes. Some of the key factors to account for are the density of the RBC themselves, the aggregation and deformability of the RBC, aggregation or movement of the fluorescing micro particles, and the settling of the RBC in the channels. All of these can be accounted for if the general guidelines laid out above are followed. There is a basic chec...

The human body contains numerous vessels with diameters less than 50 &mu;m, which are the main exchange site between blood and tissues. The study of blood flow in these vessels represents a considerable challenge due to both the scale of the measurements and the fluid properties of blood. These measurements, including the pressure gradient, the shear at the wall, and velocity profiles in arterioles and venules, are key factors linked with physiological responses. There are now unprecedented opportunities to resolve these measurement challenges, thanks to new experimental techniques at the micro scale to study the microcirculation and solve this multiscale problem.
Micro-particle image velocimetry (&mu;PIV) is a particle-based flow visualization technique that is used to evaluate velocity profiles of blood flow in microchannels via cross correlation. &mu;PIV, first developed by Santiago et al., has been used with hemorheology studies since Sugii et al. in 2001 used the technique to measure blood flow in 100 &mu;m round glass tubes 1,2. Different approaches to &mu;PIV exist. High speed cameras can be used to correlate the movement of red blood cells (RBCs), and pulsed images can be used to correlate the movement of tracer particles. Either of these options can be coupled with an upright or inverted microscope, depending on the application. In both cases, the result is a 2D velocity profile. Another approach is to use a confocal microscope to achieve 2D and 3D profiles. This method has been applied to blood 3,4,5.
Micro scale PIV has several complications when compared with macro PIV. In macro PIV the data can be limited to a single plane through sheets of light, but at the micro scale volume illumination is necessary. Volume illumination is a greater problem for the imaging of micro blood flows, as the RBCs themselves are large in comparison to the channels, and using the RBCs as the tracer particles leads to a depth of correlation (DOC) which can significantly decrease the accuracy of the cross-correlation results 6,7,8. Following Wereley et al. (1998) the DOC for a 40 &mu;m tall channel with RBC as the tracers is 8.8 &mu;m, while with a 1 &mu;m tracer particle the DOC is 6.7 &mu;m. This difference becomes more pronounced when changing channel height and magnification. Additionally, RBCs are opaque, and increasing the density of the RBCs in the flow causes imaging difficulties. Fluorescing tracer particles, first used by Santiago et al. (1998), have been advocated as a tool to decrease the influence of out-of-focus particles, when using the smallest particles possible. Using 1 &mu;m diameter fluorescing microparticles coupled with a laser is one approach that may decrease the depth of focus problem in micro blood flow imaging 10. There are several current reviews of the state of &mu;PIV technology, each of which highlights the importance of &mu;PIV to blood flow studies 11,12. Several important considerations must be taken into account when using &mu;PIV for blood. At the micro level, the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible red blood cells (RBCs), large white blood cells, platelets and other proteins suspended in a Newtonian fluid (plasma).
The velocity profiles measured here can be used to measure certain characteristics of the micro blood flows. The important factors in microhemorheology are the flow rate of the blood, the shape of the velocity profile, and the shear stress at the wall of the vessel. This information has clinical implications, as the microcirculation is the site for nutrient exchange in the body, and this exchange is shear-dependent. There are several current review studies on the state of research in the microcirculation as well 13,14,15.
Presented here is a protocol for &mu;PIV measurements of blood flows in polydimethylsiloxane (PDMS) microchannels. PDMS channels were fabricated in-house following the sources in section 1 of the protocol. Porcine blood samples were obtained from an accredited slaughterhouse and cleaned following section 2 of the protocol. All data was obtained using the LaVision MITAS &mu;PIV system, as described in section 3 of the protocol. The set-up consists of a Nd:YAG laser (New Wave Research, USA) and CCD camera (Image Intense, Lavision) controlled by a programmable triggering unit, a fluorescent microscope coupled with a stage moving in 3 axes, and a computer, in addition to a high speed camera (Dalsa 1M150, Netherlands) was added for visualization of the RBCs themselves. Both cameras are connected to a 2 port optic box (Custom by Zeiss, Germany). In typical in vivo measurements of blood flow, an upright microscope is used to track the RBCs themselves, while in typical in vitro applications an inverted microscope is used to track the tracer particles. In this unique dual set-up, the optics box allows both tracers to be imaged using the inverted microscope. Blood was introduced into the microchips via a high precision syringe pump (Nexus3000, Chemyx Inc., USA). A diagram of the system is shown in Figure 1, where the top portion of the figure represents the 140 &mu;m by 40 &mu;m rectangular channels fabricated of PDMS, and the bottom portion represents the entire system including both cameras, the laser, the syringe pump and the microscope.
Current &mu;PIV set-ups available, usually with proprietary software, include TSI, Dantec Dynamics, and LaVision. Standard cross-correlation algorithms can be achieved through numerous software options, including the MATLAB. The software is not the key, understanding what the dialogue boxes correspond to mathematically will serve the user much better. In this protocol DaVis, LaVision's proprietary software or MATLAB are utilized. The protocol is not software specific, but the menu options might be in different places in different software packages.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>poly(dimethylsiloxane) (PDMS), i.e. Sylgard-184</td>
			<td>Dow-Corning</td>
			<td>3097358-1004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E9884-100G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>poshpate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5368-10PAK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescing micro particles</td>
			<td>Microgenics/FisherSci</td>
			<td>R900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>glycerol (OPTIONAL)</td>
			<td>Sigma Aldrich</td>
			<td>G6279-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>microcentrifuge, i.e. CritSpin</td>
			<td>FisherSci</td>
			<td>22-269-291</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>syringe, i.e. 50 &mu;l Gastight</td>
			<td>Hamilton</td>
			<td>80965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>camera, i.e. Imager Intense, high speed</td>
			<td>LaVision, Dalsa</td>
			<td>Imager Intense</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>microscope, i.e. MITAS</td>
			<td>LaVision</td>
			<td>MITAS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nd:YAG laser</td>
			<td>New Wave Research</td>
			<td>Solo-II</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>syringe pump, i.e. Nexus3000</td>
			<td>Chemyx, Inc.</td>
			<td>Nexus-3000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>flexible tubing, i.e. Tygon</td>
			<td>FisherSci</td>
			<td>14-169-1A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>data processing software, i.e. DaVis</td>
			<td>LaVision</td>
			<td>DaVis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>centrifuge, i.e. Thermo Scientific CL2</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

micro-particle image velocimetry for velocity profile measurements of micro blood flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

In all figures, flow is left to right in raw images, and upwards in calculated velocity profiles. An example of the raw data obtained with blood at hematocrit H=10 flowing at 10 &mu;l/hr is shown in Figure 2. Raw data may be cross-correlated without any data processing to achieve velocity profiles. The impact of pre-processing and data processing methods is discussed by Pitts, et al., (2012b). An example of a resultant velocity profiles from data similar to Figure 2 at hematorcr...

Watch this Scientific Journal Video about Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows at JoVE.com

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

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Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.
We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro

Particle image velocimetry (PIV) is used in a wide variety of fields, due to the opportunity it provides for precisely visualizing and quantifying flows across a large spatiotemporal range. However, its implementation typically requires the use of expensive and specialized instrumentation, which limits its broader utility. Moreover, within the field of bioengineering, in vitro flow visualization studies are also often further limited by the high cost of commercially sourced tissue phantoms that recapitulate desired anatomical structures, particularly for those that span the mesoscale regime (i.e., submillimeter to millimeter length scales). Herein, we present a simplified experimental protocol developed to address these limitations, the key elements of which include 1) a relatively low-cost method for fabricating mesoscale tissue phantoms using 3-D printing and silicone casting, and 2) an open-source image analysis and processing framework that reduces the demand upon the instrumentation for measuring mesoscale flows (i.e., velocities up to tens of millimeters/second). Collectively, this lowers the barrier to entry for nonexperts, by leveraging resources already at the disposal of many bioengineering researchers. We demonstratethe applicability of this protocol within the context of neurovascular flow characterization; however, it is expected to be relevant to a broader range of mesoscale applications in bioengineering and beyond.

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro

Here we present simplified methods for fabricating transparent neurovascular phantoms and characterizing the flow therein. We highlight several important parameters and demonstrate their relationship to field accuracy.

Particle image velocimetry (PIV) is used in a wide variety of fields, due to the opportunity it provides for precisely visualizing and quantifying flows ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...