The authors acknowledge partial support for this project provided by a Collaborative Seed Grant from the Office of Research and Economic Development at UC Riverside.

1. ABS-based Sacrificial Mold Fabrication
<ol>
	<li>Design an inverse model of the desired tissue phantom using CAD software.</li>
	<li>Print the model using a 3-D printer with ABS as the building material.</li>
</ol>
2. PDMS-based Vascular Phantom Fabrication
<ol>
	<li>Mixing
	<ol>
		<li>Mix the PDMS prepolymer base and curing agent in a 10:1 ratio (by weight); a 66 g mixture provides sufficient material for the fabrication of phantoms with volumes up to 50 cm3.</li>
		<li>Place the mixture in a vacuum desiccator for 60 min to degas and minimize the bubble entrapment. Use cyclic pressurization/depressurization to facilitate bubble rupture.</li>
	</ol>
	</li>
	<li>Casting
	<ol>
		<li>Mount the printed ABS mold on a glass slide using molding putty to seal the interface.</li>
		<li>Carefully pour the PDMS mixture into the mold while trying to minimize bubble entrapment. Lingering bubbles can be manually ruptured using a needle.</li>
		<li>Cure the cast phantom at room temperature (25 &#176;C) for at least 24 h. 
		NOTE: At higher temperatures, this process can be accelerated22.</li>
	</ol>
	</li>
	<li>Demolding
	<ol>
		<li>Dissolve the ABS by submerging the phantom in acetone and sonicating for at least 15 min, using powers up to 70 W. 
		CAUTION: Acetone has a high vapor pressure at room temperature and a low flash point. Consequently, always work under a fume hood and away from potential ignition sources. Wear proper personal protective equipment (e.g., goggles or face shield, lab coat, acetone-resistant gloves).</li>
		<li>Thoroughly rinse the phantom with isopropyl alcohol and, then, DI water to remove solvent residues. 
		NOTE: PDMS swells upon exposure to acetone; however, the swelling subsides once the phantom is rinsed and dried sufficiently23.</li>
	</ol>
	</li>
	<li>Confirmation of phantom fidelity using optical microscopy
	<ol>
		<li>Using an optical microscope with an attached camera and image capture software, capture an image of a critical feature within the phantom under a magnification that maximizes the feature within the field of view.</li>
		<li>Capture an image of an appropriate calibration reticle at the same magnification.</li>
		<li>Load both images into ImageJ by dragging them onto the Toolbar.</li>
		<li>Click on the calibration reticle image to make it active and, then, select the Line tool. Using the mouse, draw a line along a feature of a known distance and select Analyze &#62; Set Scale from the ImageJ menu. 
		NOTE: In the Set Scale window, the field labeled Distance in pixels should be prepopulated with the length of the drawn line in units of pixels.</li>
		<li>Enter the length of the feature in the field labeled Known Distance, and its unit in the field labeled Unit of Length. Check the box labeled Global to apply this calibration factor to all open images.</li>
		<li>Make the image of the phantom critical feature active and use the Line tool to draw a line along a feature of interest.From the ImageJ menu, select Analyze &#62; Measure (or press Ctrl + M) to measure the length of the line.</li>
		<li>Compare the expected value against the value in the column marked Length in the Results window to confirm phantom fidelity.</li>
	</ol>
	</li>
</ol>
3. Mock Blood Solution Formulation
<ol>
	<li>Mix DI water and glycerol in a 60:40 ratio (by volume). 
	NOTE: A 100 mL volume is sufficient for the in vitro circulatory system described herein.</li>
	<li>Add 1 mL of 2.5% w/v fluorescent polystyrene bead solution (i.e., tracer particles) to the mock blood solution.</li>
	<li>Homogenize the mixture on a magnetic stir plate at 400 rpm for 10 min.</li>
</ol>
4. In Vitro Circulatory System Set-up
<ol>
	<li>Pump set-up
	<ol>
		<li>Use a wire stripper tool to cut off the DC-end plug from the AC-to-DC adapter power source.</li>
		<li>Strip the coating off the power and ground wires and connect them to the input terminal of the pulse width modulation (PWM) voltage regulator.</li>
		<li>Connect the power and ground wires from the pump&#39;s DC motor to the output terminal of the PWM voltage regulator. 
		NOTE: The PWM&#39;s seven-segment display outputs the duty cycle (0% - 100%) used to achieve a variable voltage to the DC motor.</li>
	</ol>
	</li>
	<li>Pump calibration
	<ol>
		<li>Prepare 200 mL of mock blood solution (see section 3).</li>
		<li>Place tubing from the pump inlet to the beaker holding the mock blood solution.</li>
		<li>Place tubing from the pump outlet to an empty beaker.</li>
		<li>Select a desired duty cycle set point (0% - 100%). Press the On button and start a timer.</li>
		<li>Stop the timer once the pump has transferred the entire volume of mock blood solution. Use this time to calculate the volumetric flow rate.</li>
		<li>Repeat steps 4.2.1 - 4.2.5 for at least five different duty cycle set points to establish a least-squares regression curve. 
		NOTE: A minimum of three replicate points per duty cycle set point is recommended. This relationship can be used to correlate the desired flow rate to the required PWM duty cycle.</li>
	</ol>
	</li>
</ol>
5. Video Collection
<ol>
	<li>Image calibration
	<ol>
		<li>Determine the calibration ratio for the video imaging (see section 2).</li>
	</ol>
	</li>
	<li>Apparatus set-up
	<ol>
		<li>Place the PDMS phantom on the stage of the fluorescence microscope.</li>
		<li>Connect the phantom to the gear pump and introduce the mock blood solution. 
		NOTE: Optionally, prefill the model with ethanol to facilitate full wetting; then, flush and fill it with the mock blood solution. This may be particularly beneficial for models with smaller vessels and/or blind features.</li>
		<li>Set the pump motor controller for the desired flow rate based on the pump calibration curve.</li>
		<li>Run the pump for 1 - 5 min prior to the experiment to ensure steady-state conditions.</li>
		<li>Turn on the external lamp to illuminate the field of view. Select an appropriate filter based on the excitation wavelength of the fluorescent beads.</li>
		<li>Adjust the imaging focal plane to the vessel midplane. 
		NOTE: This can be achieved by using a focal length that maximizes the imaged vessel cross-section (e.g., when using phantoms with circular vessel cross sections); and/or indexing off of a phantom feature designed to facilitate the identification of the vessel mid-plane.</li>
	</ol>
	</li>
	<li>Video recording
	<ol>
		<li>Select the video recording parameters to optimize the signal-to-noise ratio (SNR). Key parameters include exposure time, frame rate, and gain. 
		NOTE: In this protocol, we use a frame rate of 2,000 fps and a gain of 1.0. However, these parameters may vary based on the application (see the discussion section for further details).</li>
		<li>Collect the video and save it in AVI format.</li>
	</ol>
	</li>
	<li>Phantom clean-up
	<ol>
		<li>If bead-sticking is observed after an experiment, sonicate the phantom in an aqueous detergent solution using powers up to 70 W.</li>
	</ol>
	</li>
</ol>
6. Image Processing and Data Analysis
<ol>
	<li>Image preprocessing
	<ol>
		<li>Drag the saved AVI file onto the ImageJ window to import it. Select the box marked Convert to Grayscale.</li>
		<li>From the ImageJ menu, select Analyze &#62; Generate Histogram (or press Ctrl + H) to generate a histogram of image pixel intensities. Take note of the mean and standard deviation for the unprocessed image. 
		NOTE: At high frame rates, it is not unusual for the distribution to be skewed heavily toward zero (i.e., no signal).</li>
		<li>From the ImageJ menu, select Image &#62; Adjust &#62; Brightness and Contrast (or press Shift + Ctrl + H) to apply a brightness/contrast filter.</li>
		<li>On the Brightness and Contrast menu, press the Set button to define the image limits. Set the minimum value to be the mean value plus one standard deviation, and the maximum value to be the maximum intensity of the image (both based on statistics obtained in step 6.1.2). 
		NOTE: This typically eliminates all but the top 10% of the pixel intensities. The number of standard deviations may be varied depending on the desired distribution of the pixel intensities. A custom macro script for performing the intensity capping operation is provided in the Supplemental Materials.</li>
		<li>From the ImageJ menu, select Process &#62; Noise &#62; Despeckle to reduce the number of saturated pixels. 
		NOTE: This operation is necessitated by the increased potential for pixel saturation that arises during the optimization of the brightness and contrast, which can produce spurious vectors during subsequent cross-correlation.</li>
		<li>From the ImageJ menu, select Process &#62; Filters &#62; Gaussian Blur with a radius of 1.5 to reduce artifacts arising from the occasional removal of illuminated pixels in a 3 x 3 neighborhood by the prior despeckling operation.</li>
		<li>Click on the Polygon tool and, then, click on the image to outline the region of interest (ROI).</li>
		<li>From the ImageJ menu, select Edit &#62; Clear Outside to remove sensor noise in locations where no signal is expected (e.g., areas beyond the vessel wall boundary), which can decrease the overall SNR.</li>
	</ol>
	</li>
	<li>PIV calculation 
	NOTE: This portion of the protocol employs a third-party PIV plug-in for ImageJ, which relies upon Gaussian peak-fitting to enable an estimation of displacement with subpixel accuracy.
	<ol>
		<li>From the ImageJ menu, select Plugins &#62; Macros &#62; Run&#8230; and navigate to the saved macro Supplemental Code 2.ijjm to cross-correlate successive image pairs. 
		NOTE: The macro proceeds as follows. 1) A cross-correlation of the intensity field within consecutive images is first performed to determine the local displacement of advected tracer particles (i.e., the first image pair consists of the first and second images, the second image pair consists of the second and third images, etc.). 2) A two-step multipass evaluation is then performed with initial and final interrogation window sizes of 256 x 256 pixels and 128 x 128 pixels, respectively. Finally, 3) the macro performs a temporal average to further reduce the appearance of spurious vectors.</li>
	</ol>
	</li>
	<li>Normalized median test (NMT)
	<ol>
		<li>From the ImageJ menu, select Plugins &#62; Macros &#62; Run&#8230; and navigate to the saved macro Supplemental Code 3.ijjm to validate the velocity fields via the normalized median test. 
		NOTE: The macro proceeds as follows. 1) Each vector in an instantaneous vector field is first compared to its eight nearest neighbors to compute the median value. 2) The array of residual errors is then calculated as the difference between each neighboring vector and the calculated median. 3) The difference between the vector under investigation and the median neighboring vector value is then normalized by the median of the residuals. 4) This is then compared to a threshold value (typically, 0.2 pixels), which can be varied based on a priori knowledge of noise during the image acquisition. Finally, 5) a temporal average of all validated instantaneous vector fields is performed to produce a composite field, as this has been shown to increase the vector field quality24.</li>
	</ol>
	</li>
</ol>

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J., Newport, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arduino+control+of+a+pulsatile+flow+rig.">Arduino control of a pulsatile flow rig.</a> Medical Engineering and Physics. 51, 67-71 (2017).</li><li>Tsai, W., Sava&#351;, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+pumping+system+for+physiological+waveforms.">Flow pumping system for physiological waveforms.</a> Medical &amp; Biological Engineering &amp; Computing. 48 (2), 197-201 (2010).</li><li>Kato, 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=Contrast-enhanced+2D+cine+phase+MR+angiography+for+measurement+of+basilar+artery+blood+flow+in+posterior+circulation+ischemia.">Contrast-enhanced 2D cine phase MR angiography for measurement of basilar artery blood flow in posterior circulation ischemia.</a> American Journal of Neuroradiology. 23 (8), 1346-1351 (2002).</li></ol>

The authors have nothing to declare.

The protocol described herein outlines a simplified method for performing PIV studies to visualize neurovascular flows at physiologically relevant dimensions and flow conditions in vitro. In doing so, it serves to complement protocols reported by others that have also focused on simplifying the quantification of vector fields, but within very different contexts that require the consideration of far larger length scales25 or lower flow rates26,<sup class=...

PIV is widely used in experimental fluid mechanics for flow visualization and quantitative investigations of fluid motion that vary in length scale from atmospheric to microcirculatory flows1,2,3. While the specifics of its implementation can vary as widely as its applications, one aspect common to nearly all PIV studies is the use of video imaging of tracer particles seeded within the working fluid, followed by a pair-wise analysis of consecutive image frames to extract desired flow characteristics. Typically, this is accomplished by first subdividing each image frame into smaller regions termed interrogation windows. As a consequence of the random positions of the dispersed particles, each interrogation window contains a unique distribution of pixel intensities. If the window size and data acquisition rate are chosen appropriately, cross-correlation of the intensity signal in each window can be used to estimate the average displacement within that region. Finally, given that the magnification and frame rate are known experimental parameters, an instantaneous velocity vector field can be readily computed.
A major advantage of PIV over single-point measurement techniques is its ability to map vector fields across a two- or three-dimensional domain. Hemodynamic applications, in particular, have benefited from this capability, since it allows a thorough investigation of local flows, which are known to play a significant role in vascular disease or remodeling (e.g., atherosclerosis, angiogenesis)4,5,6. This has also been true for the evaluation of neurovascular flows, and the interactions thereof with endovascular devices (e.g., flow diverters, stents, intrasaccular coils), since the relevant length-scales in such applications can often span one or more orders of magnitude (e.g., from micrometer to millimeter), and device geometry and placement can significantly impact the local fluid mechanics7.
Most groups conducting PIV-based hemodynamic studies have relied on experimental set-ups that closely mimic some of the earliest investigations of stent influence on vascular flow7,8. Typically, these include a) pulsed lasers and high-speed cameras, to capture high-velocity flows; b) synchronizers, to prevent aliasing between the pulse frequency of the laser and the camera acquisition frame rate; c) cylindrical optics, to form a light sheet and, thus, minimize the background fluorescence from tracer particles above and below the interrogation plane; d) in the case of commercial turn-key systems, proprietary software packages, to perform the cross-correlation analyses. However, while some applications require the performance and/or versatility collectively afforded by these components, many others do not. Moreover, the high cost of commercially sourced tissue phantoms that recapitulate desired vascular structures can also prove limiting for many in vitro studies, particularly for phantoms with features that bridge the mesoscale regime (&#62; 500 USD/phantom). Herein, we report the development of a simplified protocol for implementing PIV for the in vitro visualization of neurovascular flows, which typically lie both spatially and temporally within the mesoscale regime (i.e., length scales ranging from submillimeter to millimeter, and velocities up to tens of millimeters/second). The protocol seeks to leverage resources already at the disposal of many bioengineering researchers, thus lowering the barrier to entry for nonexperts.
The first element of this protocol involves the use of an investment casting technique to enable the in-house fabrication of transparent, polydimethylsiloxane (PDMS)-based tissue phantoms from 3-D-printed sacrificial molds. By leveraging the increasing availability of 3-D printers in recent years, particularly those in shared/multi-user facilities (e.g., institutional facilities or public makerspaces), this methodology cuts costs significantly (e.g., &#60; 100 USD/phantom in the case presented here), while enabling a rapid turnaround for the fabrication of a wide variety of designs and geometries. In the current protocol, a fused deposition modeling system is used with acrylonitrile butadiene styrene (ABS) as the building material, and the printed part serves as a sacrificial mold for the subsequent phantom casting. Our experience has shown that ABS is well-suited for such use since it is soluble in common solvents (e.g., acetone), and it has sufficient strength and rigidity to maintain mold integrity after the removal of the support material (e.g., to prevent the deformation or fracture of diminutive mold features). In the current protocol, mold integrity is further ensured using solid printed models, although this comes at the expense of increased dissolution time. The use of hollow models may also be possible in some cases, to enhance solvent access, and thus, reduce dissolution time. However, careful consideration should be given to the effect this may have on mold integrity. Finally, while the phantoms fabricated herein are based upon idealized representations of neurovascular structures generated using a common computer-aided design (CAD) software package, the protocol is expected to be amenable to the fabrication of more complex, patient-specific geometries as well (e.g., via the use of model files generated by the conversion of clinical imaging data to the .STL file format used by most 3-D printers). Further details regarding the phantom fabrication process are provided in section 2 of the protocol.
The second element of the protocol involves the use of an open-source plug-in for ImageJ to conduct the cross-correlation analyses9. This is coupled with the implementation of a simple statistical thresholding scheme (i.e., intensity capping)10 to improve the image signal prior to cross-correlation, as well as a postcorrelation vector validation scheme, the normalized median test (NMT), to eliminate spurious vectors through a comparison of each to its nearest neighbors11. Collectively, this allows imaging to be accomplished using equipment commonly found in many bioengineering laboratories, thus eliminating the need for the acquisition of many of the costly components of typical PIV systems (e.g., pulsed laser, synchronizer, cylindrical optics, and proprietary software). Further details regarding the video collection, image processing, and data analysis are provided in sections 5 and 6 of the protocol.
Figure 1 illustrates the PIV set-up used in this protocol, which relies upon a fluorescence microscope equipped with a high-speed camera for imaging, as well as an external, continuous white-light source (i.e., metal halide lamp) for through-objective volumetric illumination. A variable-speed gear pump is used to impose the recirculating flow of a transparent mock blood solution through the neurovascular tissue phantoms. The solution is composed of a 60:40 mixture of deionized (DI) water and glycerol, which is a common substitute for blood in hemodynamic studies12,13,14, due to a) its similar density and viscosity (i.e., 1,080 kg/m3 and 3.5 cP vs. 1,050 kg/m3 and 3 - 5 cP for blood)15,16; b) its transparency in the visible range; c) its similar refractive index as PDMS (1.38 vs. 1.42 for PDMS)17,18,19,20, which minimizes optical distortion; d) the ease with which non-Newtonian behavior can be introduced, if needed, via the addition of xanthane21. Finally, fluorescent polystyrene beads are used as tracer particles (10.3 &#181;m in diameter; 480 nm/501 nm excitation/emission). While neutrally buoyant beads are desired, sourcing tracer particles with optimal fluid mechanical properties (e.g., density, size, composition) and emission wavelength can prove challenging. For example, the beads used herein are slightly less dense than the glycerol solution (1,050 kg/m3&#160;vs. 1,080 kg/m3). However, the hydrodynamic effects, thereof, are negligible, given that the duration of a typical experiment is far shorter than the time scale associated with buoyancy effects (i.e., 5 min and 20 min, respectively). Further details regarding the mock blood solution formulation and in vitro circulatory system set-up are provided in sections 3 and 4 of the protocol.

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			<td>3D printer</td>
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			<td>ABS build material&#160;</td>
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			<td>Branching perforator mold segment</td>
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			<td style="width:153px;">55205</td>
			<td>Desiccator</td>
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			<td style="width:153px;">A4514</td>
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			<td height="21" style="height:21px;width:363px;">10um Polystyrene Yellow-Green Fluorescent Particles</td>
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			<td style="width:153px;">PSF-010UM</td>
			<td>Fluorescent beads</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Phantom Miro&#160;</td>
			<td style="width:147px;">Vision Research</td>
			<td style="width:153px;">Miro M310</td>
			<td>High speed camera</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Micropump</td>
			<td style="width:147px;">Cole-Parmer</td>
			<td style="width:153px;">81101</td>
			<td>Recirculating pump</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Leica DM2000</td>
			<td style="width:147px;">Leica Microsystems</td>
			<td style="width:153px;">DM2000</td>
			<td>Fluorescent Microscope</td>
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			<td height="21" style="height:21px;width:363px;">Leica 10X Objective</td>
			<td style="width:147px;">Leica Microsystems</td>
			<td style="width:153px;">506259</td>
			<td>Objective for perforator</td>
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			<td height="21" style="height:21px;width:363px;">Leica 2.5X Objective</td>
			<td style="width:147px;">Leica Microsystems</td>
			<td style="width:153px;">11506083</td>
			<td>Objective aneurysm sac</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Leica Blue Filter Cube L5</td>
			<td style="width:147px;">Leica Microsystems</td>
			<td style="width:153px;">513840</td>
			<td>Blue filter cube</td>
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			<td height="21" style="height:21px;width:363px;">Leica EL6000</td>
			<td style="width:147px;">Leica Microsystems</td>
			<td style="width:153px;">11504115</td>
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			<td height="21" style="height:21px;width:363px;">Alconox</td>
			<td style="width:147px;">Alconox Inc</td>
			<td style="width:153px;">1104-1</td>
			<td>Detergent</td>
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			<td height="21" style="height:21px;width:363px;">ImageJ</td>
			<td style="width:147px;">NIH</td>
			<td style="width:153px;">N/A</td>
			<td style="width:361px;">Open source image analysis software 
			https://imagej.nih.gov/ij/</td>
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			<td height="21" style="height:21px;width:363px;">Particle Image Velocimetry PIV Plugin</td>
			<td style="width:147px;">Qingson Tseng</td>
			<td style="width:153px;">N/A</td>
			<td>https://sites.google.com/site/qingzongtseng/piv</td>
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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.

Figure 2&#160;illustrates the PDMS tissue phantom fabrication process. The phantoms designed herein are intended for the study of flow in idealized wide-necked, saccular, intracranial aneurysms, as well as proximal branching perforator arteries. Important additional design features include 1) a common reservoir that all vessels drain into, to ensure unencumbered fluid egress from the phantom - otherwise, droplet formation may occur at the smaller vessel outle...

Watch this Scientific Journal Video about Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro at JoVE.com

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.

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 ...

meso-scale-particle-image-velocimetry-studies-neurovascular-flows

Research

JoVE Journal

Bioengineering

8.3K Views.  University of California, Riverside. 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.

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

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 ...

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

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.

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 ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...