Name: meso-scale particle image velocimetry studies of neurovascular flows in vitro
Uploaded: 2018-12-03T13:00:00
Description: Watch this Scientific Journal Video about Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro at JoVE.com

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.</l...

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+fluid+forces+are+an+essential+epigenetic+factor+for+embryonic+cardiogenesis.">Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis.</a> Nature. 421, 172 (2003).</li><li>Ando, J., Yamamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Mechanobiology.">Vascular Mechanobiology.</a> Circulation Journal. 73 (11), 1983-1992 (2009).</li><li>Conway, D. 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(113), e51288 (2016).</li><li>Tseng, Q., 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=Spatial+organization+of+the+extracellular+matrix+regulates+cell-cell+junction+positioning.">Spatial organization of the extracellular matrix regulates cell-cell junction positioning.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (5), 1506-1511 (2012).</li><li>Shavit, U., Lowe, R. J., Steinbuck, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensity+Capping:+a+simple+method+to+improve+cross-correlation+PIV+results.">Intensity Capping: a simple method to improve cross-correlation PIV results.</a> Experiments in Fluids. 42 (2), 225-240 (2007).</li><li>Raffel, M., Willert, C. E., Werely, S., Kompenhans, J. <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: a Practical Guide. , (2007).</li><li>Kerl, H. U., 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=Implantation+of+Pipeline+Flow-Diverting+Stents+Reduces+Aneurysm+Inflow+Without+Relevantly+Affecting+Static+Intra-aneurysmal+Pressure.">Implantation of Pipeline Flow-Diverting Stents Reduces Aneurysm Inflow Without Relevantly Affecting Static Intra-aneurysmal Pressure.</a> Neurosurgery. 74 (3), 321-334 (2014).</li><li>Lieber, B. B., Livescu, V., Hopkins, L. N., Wakhloo, 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=New+Table+of+the+Refractive+Index+of+Pure+Glycerol+at+20&#176;C.">New Table of the Refractive Index of Pure Glycerol at 20&#176;C.</a> Industrial &amp; Engineering Chemistry. 26 (3), 329-332 (1934).</li><li>Cai, Z., Qiu, W., Shao, G., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+fabrication+method+for+all-PDMS+waveguides.">A new fabrication method for all-PDMS waveguides.</a> Sensors and Actuators A: Physical. 204, 44-47 (2013).</li><li>Bouillot, P., 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=Particle+imaging+velocimetry+evaluation+of+intracranial+stents+in+sidewall+aneurysm:+hemodynamic+transition+related+to+the+stent+design.">Particle imaging velocimetry evaluation of intracranial stents in sidewall aneurysm: hemodynamic transition related to the stent design.</a> PloS ONE. 9 (12), 113762 (2014).</li><li>Trager, A. L., Sadasivan, C., Lieber, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+in+vitro+hemodynamic+performance+of+new+flow+diverters+for+bypass+of+brain+aneurysms.">Comparison of the in vitro hemodynamic performance of new flow diverters for bypass of brain aneurysms.</a> Journal of Biomechanical Engineering. 134 (8), 084505 (2012).</li><li>Clauser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Plasma-Based+Fluid+for+Particle+Image+Velocimetry+(PIV):+In-Vitro+Feasibility+Study+of+Flow+Diverter+Effects+in+Aneurysm+Model.">A Novel Plasma-Based Fluid for Particle Image Velocimetry (PIV): In-Vitro Feasibility Study of Flow Diverter Effects in Aneurysm Model.</a> Annals of Biomedical Engineering. 46 (6), 841-848 (2018).</li><li>Johnston, I. D., McCluskey, D. K., Tan, C. K. L., Tracey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering.">Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering.</a> Journal of Micromechanics and Microengineering. 24 (3), 035017 (2014).</li><li>Lee, J. N., Park, C., 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=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Analytical Chemistry. 75 (23), 6544-6554 (2003).</li><li>Meinhart, C. D., Wereley, S. T., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PIV+Algorithm+for+Estimating+Time-Averaged+Velocity+Fields.">A PIV Algorithm for Estimating Time-Averaged Velocity Fields.</a> Journal of Fluids Engineering. 122 (2), 285 (2000).</li><li>Bosbach, J., K&#252;hn, M., Wagner, C., Raffel, M., Resagk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Scale+Particle+Image+Velocimetry+of+Natural+and+Mixed+Convection.">Large-Scale Particle Image Velocimetry of Natural and Mixed Convection.</a> 13th Int Symp on Applications of Laser Techniques to Fluid Mechanics. , (2006).</li><li>Meinhart, C. D., Wereley, S. T., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIV+measurements+of+a+microchannel+flow.">PIV measurements of a microchannel flow.</a> Experiments in Fluids. 27 (5), 414-419 (1999).</li><li>Lima, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+blood+flow+in+a+rectangular+PDMS+microchannel:+experimental+observations+using+a+confocal+micro-PIV+system.">In vitro blood flow in a rectangular PDMS microchannel: experimental observations using a confocal micro-PIV system.</a> Biomedical Microdevices. 10 (2), 153-167 (2008).</li><li>Kuo, C. -. C., Mao, R. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Precision+Surface+Polishing+System+for+Parts+Fabricated+by+Fused+Deposition+Modeling.">Development of a Precision Surface Polishing System for Parts Fabricated by Fused Deposition Modeling.</a> Materials and Manufacturing Processes. 31 (8), 1113-1118 (2016).</li><li>Kang, K., Oh, S., Yi, H., Han, S., Hwang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+truly+3D+microfluidic+channel+using+3D-printed+soluble+mold.">Fabrication of truly 3D microfluidic channel using 3D-printed soluble mold.</a> Biomicrofluidics. 12 (1), 014105 (2018).</li><li>Prasad, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+Image+Velocimetry.">Particle Image Velocimetry.</a> Current Science. 79 (1), 51-60 (2000).</li><li>Dellenback, P. A., Macharivilakathu, J., Pierce, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast-enhancement+techniques+for+particle-image+velocimetry.">Contrast-enhancement techniques for particle-image velocimetry.</a> Applied Optics. 39 (32), 5978-5990 (2000).</li><li>Cowen, E. A., Monismith, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+digital+particle+tracking+velocimetry+technique.">A hybrid digital particle tracking velocimetry technique.</a> Experiments in Fluids. 22 (3), 199-211 (1997).</li><li>Gruen, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+validation,+false+vectors+correction+and+derived+magnitudes+calculation+on+PIV+data.">Data validation, false vectors correction and derived magnitudes calculation on PIV data.</a> Measurement Science and Technology. 8 (12), 1493-1501 (1997).</li><li>Loudon, C., Tordesillas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+the+Dimensionless+Womersley+Number+to+Characterize+the+Unsteady+Nature+of+Internal+Flow.">The Use of the Dimensionless Womersley Number to Characterize the Unsteady Nature of Internal Flow.</a> Journal of Theoretical Biology. 191 (1), 63-78 (1998).</li><li>Drost, S., De Kruif, B. 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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 height="21" style="height:21px;width:363px;">Solidworks 2015</td>
			<td style="width:147px;">Dassault Systems</td>
			<td style="width:153px;">N/A</td>
			<td style="width:361px;">CAD Software&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Dow Corning Sylgard 184 Kit</td>
			<td style="width:147px;">Ellsworth Adhesive</td>
			<td style="width:153px;">184 SIL ELAST KIT 3.9KG</td>
			<td>PDMS Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Stratasys Dimension Elite</td>
			<td style="width:147px;">Stratasys</td>
			<td style="width:153px;">9180-00105</td>
			<td>3D printer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">P430 Model Material Cartridge</td>
			<td style="width:147px;">Stratasys</td>
			<td style="width:153px;">340-21202</td>
			<td>ABS build material&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">P400 SR Soluble Support Material Cartridge</td>
			<td style="width:147px;">Stratasys</td>
			<td style="width:153px;">340-30200</td>
			<td>Support material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">CleanStation DT3</td>
			<td style="width:147px;">PM3 Technologies</td>
			<td style="width:153px;">00-00300R</td>
			<td>Base bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Lindberg Blue M LGO Box Furnace&#160;</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td style="width:153px;">LB305745M</td>
			<td>Oven</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">21G BD PrecisionGlide Needle</td>
			<td style="width:147px;">Betcon Dickenson</td>
			<td style="width:153px;">BD 305167</td>
			<td>Branching perforator mold segment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Desiccator (Vacuum)</td>
			<td style="width:147px;">Polylab</td>
			<td style="width:153px;">55205</td>
			<td>Desiccator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Branson 1800 Utrasonic Cleaning</td>
			<td style="width:147px;">Branson</td>
			<td>CPX-952-116R</td>
			<td>Sonicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Acetone</td>
			<td style="width:147px;">Fisher Chemical</td>
			<td style="width:153px;">A9494</td>
			<td>Acetone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Isopropol Alcohol</td>
			<td style="width:147px;">Fisher Chemical</td>
			<td style="width:153px;">A4514</td>
			<td>Isopropol Alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Glycerol</td>
			<td style="width:147px;">Fisher Chemical</td>
			<td style="width:153px;">GW33500</td>
			<td>Glycerol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">10um Polystyrene Yellow-Green Fluorescent Particles</td>
			<td style="width:147px;">Magsphere</td>
			<td style="width:153px;">PSF-010UM</td>
			<td>Fluorescent beads</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Leica EL6000</td>
			<td style="width:147px;">Leica Microsystems</td>
			<td style="width:153px;">11504115</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
	</tbody>
</table>

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 ...

This method can help answer key questions in the field of bioengineering, specifically those pertaining to hemodynamics and their interactions with intervascular devices. The main advantage of this technique is that it makes use of equipment already found in most bioengineering laboratories and this can help reduce the barrier to entry for non-experts. Mix the PDMS prepolymer base in curing agent in a 10 to one ratio by weight.A 66 gram mixture provides sufficient material for the fabrication of phantoms with volumes up to 50 cubic centimeters. Place the mixture in a vacuum desiccator for 60 minutes to de-gas and minimize the bubble entrapment. Use cyclic pressurization depressurization to facilitate bubble rupture.To perform casting mount the printed ABS mold on a glass slide using molding putty to seal the interface. Carefully pour the PDMS mixture into the mold while trying to minimize bubble entrapment. Lingering bubbles can be manually ruptured using a needle.Cure the cast phantom at room temperature for at least 24 hours. A container may be used to ensure dust does not settle on the phantom while curing. To perform demolding, dissolve the ABS by submerging the phantom in acetone.Sonicate for at least 15 minutes using powers up to 70 watts. Thoroughly rinse the phantom with isopropyl alcohol and then deionized water to remove solvent residues. Using an optical microscope with an attached camera in 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.Capture an image of an appropriate calibration reticle at the same magnification. Load both images into ImageJ by dragging them onto the toolbar. 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. Set scale from the ImageJ menu. 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. 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, measure to measure the length of the line.Compare the expected value against the value in the column marked length in the results window to confirm phantom fidelity. To make the mock blood solution mix deionized water and glycerol in a 60 to 40 ratio by volume. Add one milliliter of 2.5%fluorescent polystyrene bead solution to the mock blood solution, then homogenize the mixture on a magnetic stir plate at 400 rpm for 10 minutes.Perform the in vitro circulatory system setup as described in the text protocol. Determine the calibration ratio for the video imaging as before. An acrylic sheet may be placed over the microscope stage before placing the PDMS phantom to protect the microscope from unintentional spills.To setup the apparatus, place the PDMS phantom on the stage of the fluorescence microscope. Connect the phantom to the gear pump and introduce the mock blood solution. Set the pump motor controller for the desired flow rate based on the pump calibration curve.Run the pump for one to five minutes prior to the experiment to ensure steady state conditions. If bead sticking is observed after an experiment, sonicate the phantom in an aqueous detergent solution using powers up to 70 watts. Cleanliness of the model is also critical for vector field fidelity as beads stuck to the surface of the phantom will result in interrogation windows with zero displacement.To perform image processing, drag the save AVI file onto the ImageJ window to import it. Select the box marked convert to gray scale. From the ImageJ menu select analyze, generate histogram to generate a histogram of image pixel intensities.Take note of the mean and standard deviation for the unprocessed image. From the ImageJ menu select image, adjust brightness and contrast to apply a brightness contrast filter. On the brightness and contrast menu, click 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. From the ImageJ menu select process, noise, despeckle to reduce the number of saturated pixels. Then select process, filters, gaussian blur with a radius of 1.5.This will reduce artifacts arising from the occasional removal of illuminated pixels in a 3x3 neighborhood by the prior despeckling operation. Click on the polygon tool and then click on the image to outline the region of interest. From the ImageJ menu select edit, clear outside to remove sensor noise in locations where no signal is expected which can decrease the overall signal to noise ratio.Proceed to data analysis as describe in the text protocol. Shown here is the completed phantom highlighted with dimensions as well as the particle image velocimetry or PIV region of interest. This figure shows image intensity contour plots and surface plots resulting from fluorescent beads in the perforator artery during video capture.This demonstrates the improvement in signal to noise ratio after intensity capping is performed. Shown here are the resultant vector fields obtained on raw video, after intensity capping and then again after the intensity capped vector field is validated using the normalized median test. As post processing and vector validation techniques are employed the vector field becomes more uniform and more closely resembles the expected profile for flow in a circular channel.While attempting this procedure it's important to focus on minimizing the noise in the acquired signal itself. While this protocol outlines software based techniques to mitigate these aberrations, care should be taken at each step to reduce their occurrence. Don't forget that working with acetone can be extremely hazardous and you should always wear personal protective equipment as well as work under a fume hood away from any ignition sources while performing this procedure.

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

Research

JoVE Journal

Bioengineering

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 ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...