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. Oxygenated plasma treatment is a common option.
Zhou, Ellis, and Voelcker (2009) give a review of surface treatments and how they affect PDMS. These results are for water however, and blood has different properties. A study on what that surface treatment means for blood has been done by Pitts et al. (2012a).
For the results presented here, microchip surfaces and the glass slides they were bonded to were both exposed to oxygenated plasma for 45 seconds and then pressed together firmly resulting in a permanent bond.
2. Blood Preparation
<ol>
	<li>Collect blood samples from an accredited facility. For example porcine blood can be used with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Add 1 g EDTA with 4 ml of water, and then add the solution to 1 L of whole blood, mixing gently 10x.</li>
</ol>
When picking up blood samples, allow them to cool slowly to RT. Blood samples should be fresh, and ideally used the day they are collected to conserve the rheological properties of the blood.
Samples may be refrigerated once at RT; however whole blood without anticoagulant will be unusable after refrigeration. Depending on the anticoagulant, RBCs can be kept refrigerated for up to 42 days, and whole blood can be kept refrigerated for 35 days, but contamination would be possible in a non-medical facility 21.
Additionally, be careful of the collection method with bovine or porcine samples, and avoid contamination by bone marrow.
<ol start="2">
	<li>Centrifuge the blood samples 3x at 3,000 rpm for 10 min each time. After the first centrifugation, remove the plasma and buffy coat, discard. It is generally easiest to remove the plasma first, and then discard or keep for future use, then to remove the buffy coat alone.</li>
</ol>
Introduce the pipette slowly, so as to not mix the buffy coat back into the blood. After removing the buffy coat, add about 20 ml of phosphate buffered saline (PBS) and mix gently, recentrifuge. Repeat last step. After the third centrifugation, remove the PBS and remaining buffy coat, discard.
It is also possible to use the native plasma instead of PBS if you wish to keep the aggregating properties of the blood. Make sure to not mix any buffy coat or white blood cells into the plasma.
<ol start="3">
	<li>Take cleaned red blood cells (RBCs) and suspend in PBS or plasma at desired concentrations (hematocrits). Check hematocrits with a microcentrifuge (CritSpin FisherSci, USA).</li>
</ol>
Blood at 10 to 20% hematocrit should be easier to visualize than 40 or 50% (physiological hematocrit). In the microcirculation, the blood is generally half the hematocrit of the macro circulation, so H=20 is adequate 15.
<ol start="4">
	<li>Add fluorescing tracer particles at the desired concentration to the blood samples. A general rule is to have 10 particles in a correlation window, which can be calculated ahead of time.</li>
</ol>
For example, 30 &mu;l of particles are added to 1 ml of blood solution for the set-up depicted in the video. Alternatively, the RBCs themselves could be fluorescently tagged.
<ol start="5">
	<li>Additionally, make a calibration solution with water and fluorescing particles. It will be necessary to calibrate the system with a Newtonian solution.</li>
</ol>
For example, when using a 10X objective with a channel on the scale of 100 &mu;m, a suitable concentration is 300 &mu;l of particles mixed with 15 ml of distilled water. Another option is to use glycerol diluted with distilled water to a viscosity of 3cP, which is closer to blood's macro viscosity.
3. &mu;PIV Measurements
<ol>
	<li>Laser safety: Check temperature and humidity. Wear appropriate laser safety glasses. Close the laser curtain or turn on the laser sign on the lab door on to prevent people not protected from being exposed.</li>
	<li>Procedure with Davis software is shown in the video. For more details refer to the user manual of a specific system.</li>
	<li>Before taking data, the camera scale needs to be calibrated using a micrometer.</li>
	<li>To reduce the compliance of the system, use the shortest amount of tubing and the smallest rigid syringe possible, such as a 15 or 50 &mu;l Hamilton Gastight syringe. To reduce leakage of blood or air intake into the system, seal the tubing to the syringe and to the chip. This can be done with glue, PDMS or Vaseline (being careful not to contaminate the blood).</li>
</ol>
To fill the micro-syringe with water laced with tracer particles use backflow method because the volume of the micro-syringe is usually much smaller than the volume to fill.
Our backflow method consists to fill the micro-syringe and channel together via the output of the channel using a secondary plastic syringe. For that, first remove the micro-syringe plunger, then push the liquid using the classical plastic syringe attached to the outlet of the channel, let the liquid flow out the micro-syringe until all microbubbles are out of the micro -syringe, tubing or chip, finally put the plunger back, and unplug the plastic syringe. The presence or absence of microbubbles can be verified with a microscope.
<ol start="5">
	<li>Level the syringe pump to achieve horizontal tubing. Program the syringe pump to desired flow rate.</li>
</ol>
Be aware of the possible transitory effects, like the "bottleneck effect", for rigid system or the compliance of the system that modify the actual flow rate. Characteristic times of a system can be estimated as a function of the materials and dimensions of the system. (Chapter 2, pp. 77-81, Tabeling, 2005)
<ol start="6">
	<li>Place microchip on microscope stage and start the pump. Use the water with particles to calibrate the system to the middle velocity profile and calibrate the dT (the time between pulsed images).</li>
</ol>
The particles should move between 5 and 10 pixels between frames for a good correlation 11. When calibrating, be careful to measure in the middle of the channel. The focus plane in the middle has the highest velocity 8.
If using a round channel, be careful of shadowing effects.
A sample image is shown in Figure 2, using a pulsed camera, whereas the top image is the first pulse and the bottom image is the second pulse. It can be seen in the figure that the brightest points (fluorescing particles) are the most in-focus.
<ol start="7">
	<li>OPTIONAL: By taking data at different heights a 3D velocity profile can be reconstructed by adding the different vectors into a single image. The video presents a routine that was developed for the automation of the process.</li>
	<li>When taking data with the blood, fill syringe with desired quantity of blood in the same method as the water. Program the syringe pump to the desired flow rate, and take desired data. An example of raw images obtained is presented Figure 2. Be careful of RBC sedimentation as well. Refilling the syringe every run or every other run will reduce the settling of the RBC.</li>
	<li>After acquiring images, cross correlation is performed on the image pairs to obtain velocity vector fields between images. It is important to have good images to have a good correlation. Before correlating, pre-processing can be done to remove background noise.</li>
</ol>
Cross correlation is done between windows in the adjacent images, which can be varied in size and shape to affect the correlation. After cross correlation, image post processing can be done to remove erroneous vectors or outliers.
A detailed description of the different available options and their accuracies can be found in Pitts et al. (2012b), where the best processing for blood was found to be the method of "image overlapping" with windows extended in length and aligned with the flow. These operations can be done manually in a program like MATLAB, or within the software package with your system. The software is not important, the math behind the operations is more important and each operation can affect the accuracy of the final velocity profile.
The resulting vectors can be averaged in space across the channel to obtain instantaneous velocity profiles, and then averaged again in time to obtain an average, representative velocity profile. Kloosterman et al. (2011) give an explanation of the error in &mu;PIV measurements where the depth of field is large. Discussions on the error of the data processing presented here can be found in Pitts et al. (2012b) for the pulsed measurements and Chayer et al. (2012) for the high speed measurements.
Examples of velocity profiles are presented in Figures 3 and 4. Figure 3 presents a single velocity profile for the centerline of a 10 &mu;l/hr flow of RBC at H=10 in a 140 &mu;m wide channel.
This single profile is achieved by averaging the correlated vectors across the channel to get a velocity profile, and then averaged in time to get a representative measurement. In this case, 100 pairs were averaged in time across the field of view.
An example of the 3D reconstructed profile taken for a water calibration flow in a 100 &mu;m square channel with a programmed flow rate of 30 &mu;l/hr is found in Figure 4. In Figure 4, the profiles are measured at 1 &mu;m intervals.
<ol start="10">
	<li>OPTIONAL: In the depicted set-up high speed images can be taken at the same conditions by switching cameras (and data acquisition systems). This can be useful for visualizing the cell-free layer in the channel, and for quantifying aggregation. The data analysis is slightly different (Chayer et al., 2012).</li>
</ol>
Examples of white light images with and without RBC aggregation are presented in Figure 5 at H=20. The top images shows H=20 in PBS, which removes the ability of the RBC to aggregate, at 1 &mu;l/hr. The bottom image is H=20 in native plasma at 0.5 &mu;l/hr. In the bottom image, aggregation is visible.
<ol start="11">
	<li>Shut down the system in a safe manner when measurements have been completed. Use proper laser safety procedures until laser power source is completely shut down.</li>
</ol>

<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. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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. 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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>

The authors have nothing to disclose.

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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17.1K Views.  University of Ottawa. Micro-particle image velocimetry (μPIV) 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.

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

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 ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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  &amp;#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 ...