Studies presented here were supported by funding from the National Institutes of Health (NIH U01 GM111243 and NIH NIDDK P30 DK079312). Intravital microscopy studies were conducted at the Indiana Center for Biological Micros&#173;copy. We thank Dr. Malgorzata Kamocka for technical assistance with microscopy.

<ol>
	<li>Shen, 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=Subclinical+Vascular+Dysfunction+Associated+with+Metabolic+Syndrome+in+African+Americans+and+Whites.">Subclinical Vascular Dysfunction Associated with Metabolic Syndrome in African Americans and Whites.</a> The Journal of clinical Endocrinology and Metabolism. 100 (11), 4231-4239 (2015).</li><li>Sherman, I. A., Pappas, S. C., Fisher, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+microvascular+changes+associated+with+development+of+liver+fibrosis+and+cirrhosis.">Hepatic microvascular changes associated with development of liver fibrosis and cirrhosis.</a> American Journal of Physiology-Heart and Circulatory Physiology. 258 (2), 460-465 (1990).</li><li>Zafrani, L., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculation+in+Acute+and+Chronic+Kidney+Diseases.">Microcirculation in Acute and Chronic Kidney Diseases.</a> American Journal of Kidney Diseases. 66 (6), 1083-1094 (2015).</li><li>Nielsen, R. B., 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=Capillary+dysfunction+is+associated+with+symptom+severity+and+neurodegeneration+in+Alzheimer's+disease.">Capillary dysfunction is associated with symptom severity and neurodegeneration in Alzheimer's disease.</a> Alzheimer's &amp; Dementia. 13 (10), 1143-1153 (2017).</li><li>Houben, A. J. H. M., Martens, R. J. H., Stehouwer, C. D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Microvascular+Function+in+Humans+from+a+Chronic+Disease+Perspective.">Assessing Microvascular Function in Humans from a Chronic Disease Perspective.</a> Journal of the American Society of Nephrology. 28 (12), 3461-3472 (2017).</li><li>Clemens, M., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Sinusoidal+Perfusion:+In+Vivo+Methodology+and+Control+by+Endothelins.">Regulation of Sinusoidal Perfusion: In Vivo Methodology and Control by Endothelins.</a> Seminars in Liver Disease. 19 (04), 383-396 (1999).</li><li>Uhlmann, S., Uhlmann, D., Spiegel, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hepatic+microcirculation+by+in+vivo+microscopy.">Evaluation of hepatic microcirculation by in vivo microscopy.</a> Journal of investigative surgery : the official journal of the Academy of Surgical Research. 12 (4), 179-193 (1999).</li><li>Dunn, K. W., Sutton, T. A., Sandoval, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+imaging+of+renal+function+by+multiphoton+microscopy.">Live-animal imaging of renal function by multiphoton microscopy.</a> Current protocols in cytometry. , (2012).</li><li>von Diezmann, A., Shechtman, Y., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Localization+of+Single+Molecules+for+Super-Resolution+Imaging+and+Single-Particle+Tracking.">Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking.</a> Chemical Reviews. 117 (11), 7244-7275 (2017).</li><li>Huang, Z. -. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization-based+super-resolution+microscopy+with+an+sCMOS+camera.">Localization-based super-resolution microscopy with an sCMOS camera.</a> Optics Express. 19 (20), 19156 (2011).</li><li>Clendenon, S. G., 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+simple+automated+method+for+continuous+fieldwise+measurement+of+microvascular+hemodynamics.">A simple automated method for continuous fieldwise measurement of microvascular hemodynamics.</a> Microvascular Research. 123, 7-13 (2019).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Liu, Z. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale+space+approach+to+directional+analysis+of+images.">Scale space approach to directional analysis of images.</a> Applied Optics. 30 (11), 1369 (1991).</li><li>Linkert, M., 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> The Journal of cell biology. 189 (5), 777-782 (2010).</li><li>Sherman, I. A., Pappas, S. C., Fisher, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+microvascular+changes+associated+with+development+of+liver+fibrosis+and+cirrhosis.">Hepatic microvascular changes associated with development of liver fibrosis and cirrhosis.</a> American Journal of Physiology-Heart and Circulatory Physiology. 258 (2), 460-465 (1990).</li><li>Babbey, C. M., 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=Quantitative+intravital+microscopy+of+hepatic+transport.">Quantitative intravital microscopy of hepatic transport.</a> IntraVital. 1 (1), 44-53 (2012).</li><li>Dunn, K. W., Ryan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+quantitative+intravital+multiphoton+microscopy+to+dissect+hepatic+transport+in+rats.">Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats.</a> Methods. 128, 40-51 (2017).</li><li>Ryan, 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=Intravital+Multiphoton+Microscopy+with+Fluorescent+Bile+Salts+in+Rats+as+an+In+Vivo+Biomarker+for+Hepatobiliary+Transport+Inhibition.">Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition.</a> Drug metabolism and disposition: the biological fate of chemicals. 46 (5), 704-718 (2018).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Glomerular+Permeability+of+Fluorescent+Macromolecules+Using+2-Photon+Microscopy+in+Munich+Wistar+Rats.">Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats.</a> Journal of Visualized Experiments. (74), e50052 (2013).</li><li>Chhatbar, P. Y., Kara, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+blood+velocity+measurements+with+a+hybrid+image+filtering+and+iterative+Radon+transform+algorithm.">Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm.</a> Frontiers in Neuroscience. 7, 106 (2013).</li><li>Drew, P. J., Blinder, P., Cauwenberghs, G., Shih, A. Y., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+particle+velocity+from+space-time+images+using+the+Radon+transform.">Rapid determination of particle velocity from space-time images using the Radon transform.</a> Journal of computational neuroscience. 29 (1-2), 5-11 (2010).</li><li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (26), 15741-15746 (1998).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy+Imaging+of+the+Liver+following+Leishmania+Infection:+An+Assessment+of+Hepatic+Hemodynamics.">Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.</a> Journal of visualized experiments : JoVE. (101), e52303 (2015).</li><li>Hoshikawa, 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=Dynamic+Flow+Velocity+Mapping+from+Fluorescent+Dye+Transit+Times+in+the+Brain+Surface+Microcirculation+of+Anesthetized+Rats+and+Mice.">Dynamic Flow Velocity Mapping from Fluorescent Dye Transit Times in the Brain Surface Microcirculation of Anesthetized Rats and Mice.</a> Microcirculation. 23 (6), 416-425 (2016).</li><li>Sironi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+flow+mapping+in+complex+vessel+networks+by+single+image+correlation.">In vivo flow mapping in complex vessel networks by single image correlation.</a> Scientific reports. 4 (1), 7341 (2014).</li><li>Kamoun, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+RBC+velocity,+flux,+hematocrit+and+shear+rate+in+vascular+networks.">Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks.</a> Nature Methods. 7 (8), 655-660 (2010).</li></ol>

The authors report no competing interests.

There are multiple critical steps in this protocol. First, minimization of motion during intravital imaging of the liver is essential for generating movies that are usable for capillary flow analysis using STAFF. Due to the proximity of the diaphragm, short periods of respiration-induced motion occur, with the secured liver returning to its initial position after each breath. Securing the surgically exposed liver against the coverslip-bottomed dish using gauze, then imaging from below using an inverted microscope serves ...

The microvasculature plays a critical role in physiology, ensuring effective perfusion of tissues under changing conditions. Microvascular dysfunction is associated with myriad conditions including long-term cardiovascular morbidity and mortality, development of dementia, and disease of both liver and kidney and thus is a key factor of interest in a broad range of biomedical investigations1,2,3,4,5. While multiple techniques have been used to evaluate tissue perfusion, only intravital microscopy enables data collection at the temporal and spatial resolution necessary to characterize blood flow at the level of individual capillaries.
Microvascular flow can be visualized in fluorescence microscopy either by the movement of fluorescent microspheres or by the movement of red blood cells against the background of membrane-impermeant fluorescent markers (e.g., fluorescently-labeled dextran or albumin)6,7. Microvascular flow can be imaged in superficial cell layers using widefield microscopy, or at depth using either confocal or multiphoton microscopy. However, capillary flow rates are such that the passage of red blood cells cannot generally be captured at speeds less than 60 frames/s. Since most laser scanning confocal and multiphoton microscopes require 1&#8211;5 s to scan a full image field, this speed can generally be accomplished only by limiting the field of view, sometimes to a single scan line8. The process of limiting measurements to selected capillary segments (1) has the potential to introduce selection bias and (2) makes it impossible to capture spatial and temporal heterogeneity in the rates of capillary blood flow. In contrast, images of capillary networks can be collected at speeds exceeding 100 fps using widefield digital microscopes equipped with scientific complementary metal oxide semiconductor (sCMOS) cameras9,10. These inexpensive systems, common in typical biomedical laboratories make it possible to image microvascular flow across entire two-dimensional networks, essentially continuously. The problem then becomes one of finding an analysis approach that is capable of extracting meaningful quantitative data from the massive and complex image datasets generated by high-speed video microscopy.
To enable analysis of full-field flow data we have developed STAFF, novel image analysis software that can continuously measure microvascular flow throughout entire microscope fields of image series collected at high speed11. The approach is compatible with a variety of different experimental systems and imaging modalities and the STAFF image analysis software is implemented as a macro toolset for the FIJI implementation of ImageJ12. The underlying principle used here to visualize microvascular flow is that first, some contrast must be provided to be able to image the red blood cells within capillaries. In our studies, contrast is provided by a bulk fluorescent probe that is excluded by the red blood cells. The velocity of flow can then be quantified from the displacement of the red blood cells that appear as a negative stain within the fluorescently labeled plasma in images collected at high speed from a living animal8. We then use STAFF to make plots of distance along each capillary segment over multiple intervals of time called kymographs, then detect the slopes present in the kymographs13, and from those slopes calculate the rates of microvascular flow. The approach can be applied to images collected from any capillary bed that can be accessed for imaging. Here we describe the application of IVM and STAFF to studies of blood flow in the liver.

<table><tbody><tr><td>#5 forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>Dumont #5 Inox Forceps</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Labs</td><td></td><td>male 9-12 weeks old</td></tr><tr><td>Cannula</td><td>Instech</td><td>BTPE-10</td><td>Polyethylene Tubing 0.011 x 0.024 inches</td></tr><tr><td>CMOS camera</td><td>Hamamatsu</td><td>C11440-42U30</td><td>4.0LT Scientific CMOS</td></tr><tr><td>Coverslip-bottomed dish</td><td>Electron Microscopy Sciences</td><td></td><td>WillCo Dish glass bottom GWST5040</td></tr><tr><td>Dissecting scissors</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Fiji ImageJ Image analysis software</td><td></td><td></td><td>https://fiji.sc/ ; https://downloads.imagej.net/fiji/Life-Line/fiji-win64-20170530.zip</td></tr><tr><td>Fluorescein dextran</td><td>Thermo Fisher, Invitrogen</td><td>D1822</td><td>Dextran, Fluorescein, 70,000 MW, Anionic, Lysine Fixable</td></tr><tr><td>Gauze sponge</td><td>Fisher</td><td>22-415-504</td><td>2x2 inch Dukal sterile gauze sponges</td></tr><tr><td>Heating pad</td><td>Reptitherm</td><td>RH-4</td><td>between mouse and stage</td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>000732-500-000U</td><td>over mouse</td></tr><tr><td>Inverted epifluorescence microscope</td><td>Nikon</td><td></td><td>Nikon TiE inverted microscope</td></tr><tr><td>Isis Rodent electric shaver</td><td>Braun Aesculap</td><td>GT420</td><td></td></tr><tr><td>Isofluorane</td><td>Abbott GmbH</td><td>PZN4831850</td><td></td></tr><tr><td>Luer stub adapter</td><td>Fisher</td><td>14-826-19E</td><td>Catheter adapter</td></tr><tr><td>Micro scissors</td><td>Castro Viejo</td><td></td><td></td></tr><tr><td>Microscope objective</td><td>Nikon</td><td></td><td>Plan Fluor 20x, NA 0.75 water immersion</td></tr><tr><td>Needle</td><td>Fisher</td><td></td><td>30 G x1/2&quot;</td></tr><tr><td>Needle holder</td><td>Olsen-Hegar</td><td></td><td></td></tr><tr><td>Objective heater</td><td>BioScience Tools</td><td>MTC-HLS-025</td><td>Temperature controller with objective heater</td></tr><tr><td>Rectal thermometer</td><td>Braintree Scientific, INC</td><td>TH-5A</td><td>Mouse Body Temperature monitoring</td></tr><tr><td>STAFF macros</td><td></td><td></td><td>https://github.com/icbm-iupui/STAFF</td></tr><tr><td>Suture string</td><td>Harvard Bioscience</td><td>723288</td><td>silk black suture, 6-0, spool</td></tr></tbody></table>

spatial temporal analysis of fieldwise flow in microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

STAFF analysis generates a complete census of microvascular velocities across entire microscope fields over periods of time extending from seconds to minutes. Representative results are presented in Figure 1, Figure 2, Figure 3, and Figure 4. Figure 1 shows an example of a time series of the microvascular network in the liver of a mouse, the generation of the skeletonized i...

Watch this Scientific Journal Video about Spatial Temporal Analysis of Fieldwise Flow in Microvasculature at JoVE.com

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

spatial-temporal-analysis-of-fieldwise-flow-in-microvasculature

Research

JoVE Journal

Bioengineering

5.8K Views.  Indiana University. Aangezien de microvasculaire stroom varieert tussen haarvaten en veranderingen in een tijdsloop van seconden, is een methode voor de continue kwantificering van de stroom over hele velden nodig. Het personeel maakt een bijna continue meting van de stroomsnelheden over hele microscoopvelden mogelijk, binnen enkele uren, die anders maanden zouden duren om handmatig te verkrijgen. Hoewel deze procedures eenvoudig zijn, zal visuele demonstratie nieuwe gebruikers aanzienlijk helpen om voor het eer...

Video: Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

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 ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Medicine

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Developmental Biology

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

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

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

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

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

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Samenvatting

Meer video's verkennen