Velocity Measurement of Blood Flow in a Microtube Using Micro PIV System

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1 June 3-5, 23, Chamonix, France. F484 Velocity Measurement of Blood Flow in a Microtube Using Micro PIV System R. Okuda 1*, Y. Sugii 1 and K. Okamoto 1 1 Nuclear Engineering Resarch Laboratory, University of Tokyo, Japan. KEYWORDS: Main subject(s): Bio-Fluid Flow, Fluid: Hydrodynamics, Visualization method(s): Particle Image Velocimetry (PIV), Other keywords: Blood flow, Micro PIV system, Micro round tube, ABSTRACT : Biophysical behavior of blood in the vasculature is of importance in microcirculation researches. Especially, rheological properties of blood flow have been studied using in vivo and in vitro experiments. This paper describes flow characteristics of in vitro blood flow. In order to obtain velocity distributions with high spatial resolution, a micro PIV technique, which consists of a fluorescent microscope, double-pulsed YAG laser and cooled CCD camera, was applied to in vitro blood flow experiment through a micro round tube of a diameter 1 µm. Velocity distributions with spatial resolutions of 5.9 x.73 µm were obtained even near the wall region. The results showed typical characteristics of Non-Newtonian fluid that the axial velocity profiles of blood flow were blunt around the center region of a tube. Moreover, blood flow velocity and water flow profiles were compared in order to clarify blood characteristic. 1. Introduction Microcirculation maintains tissue and organs by delivering oxygen and materials. Biophysical behavior of blood in the vasculature is of importance in microcirculation researches. Rheological properties of RBCs (Red Blood Cells) have been studied using in vivo and in vitro experiments. A number of techniques have been proposed for measuring a velocity distribution, such as the laser Doppler velocimetry (Cochrane et al, 1981), the dual slit method (Wayland et al, 1967) and so on. However, these conventional methods were not suitable for microcirculation analysis respect to measurement accuracy and spatial resolution. Several techniques based on microscopic video image have also been proposed, in which a velocity vector was usually obtained by tracking a RBC in two successive images. These techniques have limited spatial and temporal resolution depending on the performance. Particle Image velocimetry (PIV) is a quantitative method for measuring velocity fields, instantaneously. In order to improve measurement accuracy and spatial resolution, the PIV technique, such as the cross-correlation method, particle tracking method and iterative correlation method, has been developed for macroscopic fluid system. Recently, so-called micro PIV techniques, using a microscope and a CCD camera, have been developed for applications of micro-fluidic devices. The authors have applied a micro PIV technique, which consisted of an intravital microscope, continuous * Corresponding author: Remi Okuda, Tokai-mura, Ibaraki, , Japan okuda@utnl.jp 1 Copyright 23 by PSFVIP-4

2 June 3-5, 23, Chamonix, France. F484 halogen lamp and high-speed CCD camera at 1 frame/sec, to in vivo blood images of mesenteric arterioles in a living rat (Sugii et al, 21) and have also improved the accuracy of RBCs velocity by taking mesentery motion into account (Sugii et al, 22). The results showed complex flow features, such as Non-Newtonian effect, two phase flow, pulsatile flow and so on. In order to simplify these complicated flow characteristics, it is important to do model experiments, such as in vitro experiment using a microtube, instead of in vivo experiment through an actual blood vessel. Gijisen et al, (1999) experimentally and numerically studied the influence of the non-newtonian properties of blood flow by flowing non-newtonian medium through a curved tube. Koutsiaris et al, (1999) measured velocity field of suspensions seeded with particle of diameter 1 µm flowing inside glass capillaries with internal diameter of 2 µm, using PIV technique. Meinhert et al, (1999) used a pulsed laser and a cooled CCD camera to measure a velocity field in a 3 µm x 3 µm x 25 mm rectangular microchannel seeded with 2 nm diameter particle. The author obtained velocity distribution of blood flow seeded with diameter of 3 µm through a micro round tube with inner diameter of 1 µm (Okuda et al, 22). However, since thickness of plasma layer is approximately 2-3 µm and almost equivalent to a diameter of fluorescent particle, the velocities especially in the plasma layer can t be obtained with high measurement accuracy. Moreover, the obtained velocity near a wall in vivo previous experiment of mesenteric arterioles in a living rat does not have enough accuracy due to small particle density. Therefore, it is necessary to improve measurement accuracy even near the plasma layer, in order to investigate mechanical interactions between blood cells and endothelium. In this paper, in order to basically investigate flow characteristics of blood flow compared with water flow respect to microscale flow, micro PIV technique was applied to in vitro blood flow experiment through a micro round tube. The micro PIV technique consisted of fluorescent microscope using a double-pulsed laser and a cooled CCD camera. Velocity distribution of blood flow seeded with fluorescence particle was obtained. 2. Experimental setup Microcirculation is composed of arterioles, capillaries and venules, which have diameters of 5 to 5 µm. Blood consisted of blood cell components such as a RBC with amount to 4-45% on volume rate, a white blood cell and a platelet with amount to less than.5 % on volume rate, and liquid component such as plasma. Fig.1 shows a typical stationary RBC image illuminated by halogen lamp and flowing RBC image illuminated by pulse laser. RBCs have round form as biconcave disks of a diameter about 6 µm and thickness of about 2.5 µm and deformed like a parachute shape caused by shear stress. Blood density ρ is about 1.5 g/cm 2 and apparent viscosity of blood µ is about cp, which depends on hematocrit, shear rate and so on. The blood viscosity is increased markedly at low flow shear-rates due to RBC s aggregation, while it remains almost constant at high flow shearrates. Blood flow in microvessels shows flow features that RBCs drift to the central axis of vessel (axial drift) and that a cell-free layer is formed along the vascular wall (plasma layer). In vivo blood flow shows complicated flow state due to geometry of blood vessel and pulsation by heart beat and so on. One of the reasons is why a blood vessel of living body complicated shape due to asymmetry and roughness. In vitro experiments using microtube simulated arteriole played a major role to simplify complicated rheological flow features. Since certain factors, such as vessel geometry, flow rate, hematocrit and so on, can be controlled, in vitro experiments was helpful in investigating basically biophysical behavior. 6 µm 6 µm (a) Stationary condition Fig.1 Red Blood Cell (b) Flowing condition 2 Copyright 23 by PSFVIP-4

3 June 3-5, 23, Chamonix, France. F484 In the experiments, in order to investigate blood flow characteristic, in vitro experiments using an inner flow of micro round tube, which has uniform diameter and smooth surface was carried out. Fig.2 illustrates a schematic view of in vitro blood flow experimental set up. A microtube with inner diameter 1 µm and outer diameter 3 µm was dipped in the water. Normally in the case of round tube experiments, a reference light was refracted by curvature due to different refractive index between fluid through the tube material made of glass or plastic and so on. It was difficult to observe an inner flow and especially near the wall. In order to improve the problem, the microtube made of FEP (Fluoeinated Ethylene Polymer), which have approximately equivalent refractive index to that of water 1.33, was used. The micro PIV system consisted of a double-pulsed Nd:YAG laser ( λ = 532 nm) and a cooled CCD camera with resolution of 128 x 124 pixels, 12 bit per pixel instead of high speed camera. Fluorescent particles in the observed region were illuminated from downside of the microscope stage. Absorbing green light by laser beam (peak wavelength, λ = 542 nm), fluorescent dye on particles with a diameter of.5 µm emits the red light (peak wavelength, λ = 612 nm). The fluorescent particle images were captured through a microscope equipped an optical colour filter (λ = 55 nm) and water immersion 6x objective lens with long working distance WD = 2. mm, a numerical aperture NA =.9. A center plane in depth direction can be observed because of long working distance. Illuminating double pulsed laser just before camera frame close and just after next frame open, two successive images in very short time interval can be obtained. By controlling time interval between double-pulsed laser illuminations, a particle displacement in the image plane was optimised. Two kinds of working fluid, water flow and blood flow, were used for investigating blood flow characteristic. In the case of the water flow ion-exchanged water seeded with fluorescent particle, and in the case of the blood flow a blood of rabbit heparinized for preventing blood coagulation and also seeded with fluorescent particle were taken. Flow rate was set to be constant using a syringe pump equipped a micro syringe with 1 µl capacity. Two cases with different flow rate were settled as 2 µl/h and 1 µl/h on water flow case and blood flow case, respectively. The average velocities were set to about.7 mm/sec and 3.5 mm/sec, based on Poiseuille s low theoretically. Control Unit Cooled Camera CCD Colour filter Intravital Microscope microtube PC Double-pulsed mirror Nd:YAG laser Fig.2 Experimental setup 3. Results and discussion Fig.3 shows a blood flow image in a micro tube illuminated by a halogen lamp. The observed region was 242 x 194 µm in size, with each pixel representing a.18 x.18 µm area. The focal plane was set around the center of a microtube in the depth direction. Fluorescent particles can be observed as small points near the wall region. Fluorescent particle with enough smaller size than cells was flowed even very close to the wall region. Contrary, RBC can t be observed near the wall region because physical forces tend to make RBCs migrate toward the center region of the tube. A cell-free layer, called a plasma layer, can be observed near the wall. The long path-line both fluorescent particle and RBCs around the center region can be observed due to long exposure time of the camera compared with particle and cell displacement. 3 Copyright 23 by PSFVIP-4

4 Home Program Go to Previews View Proceedings of PSFVIP-4 June 3-5, 23, Chamonix, France. F484 Fig.4 shows a fluorescent particle image of blood flow illuminated by a double-pulsed laser. A fluorescent particle with diameter of.5 µm was used. A fluorescent particle was clearly observed as bright point sources and randomly distributed within the tube. Conversely, flowing RBCs can t be observed in the flow image. The wall interfaces around the center plane in depth direction were indistinctly recognized in upper and lower of the image. Since the refractive index of tube corresponds to that of water, particle around the wall can be clearly observed X Position [ µm ] 2 Fig.3 Blood flow image illuminated by halogen lamp X Position [ µm ] 2 Fig.4 Fluorescent particle image illuminated by double-pulsed laser Fig. 5 shows the time-averaged velocity distributions of water flow case and blood flow case through the microtube calculated using the highly PIV technique (Sugii et al., 2), respectively. An interrogation window of 64 x 8 pixels was taken with 5% overlap, corresponding to a spatial resolution of 5.9 x.74 µm. 19 velocity vectors were obtained in the cross-section. The velocity vectors in the horizontal direction were thinned out to display clearly. The flows in both cases look like typical laminar flow. The velocities outside of the flow were approximately zero. The velocity vectors very close to the wall were measured because of eliminating refraction effects. Since the flows were fully developed, wall-normal components were close to zero. The spatial fluctuation of velocity profiles in the cross-sections in the case of blood flow was larger than that in the case of water flow. It was 4 Copyright 23 by PSFVIP-4

5 June 3-5, 23, Chamonix, France. F484 also observed that the velocity profiles around the center of tube in the case of blood flow became blunt. Fig.6 shows the time-averaged axial velocity profiles and temporal variances of water flow case and blood flow case in a cross section at x = 16 µm. Red solid lines of both cases represent axial velocity profiles at average velocity 3.5 mm/sec, which was obtained using flow rate 2 µl/h and area of cross-section of microtube, and red dot lines do temporal variances. Blue solid and dot lines also show axial velocity profiles and temporal variances at average velocity.7 mm/sec, flow rate 1 µl/h. The velocity and variance were normalized with each maximum velocity. The maximum velocities in the case of water flow at two flow rates were obtained as approximately 1.6 mm/sec and 8.4 mm/sec. The maximum velocities in the case of blood flow were obtained as approximately 1.3 mm/sec and 8.6 mm/sec, respectively. The velocity of all profiles became maximum around the center of the tube, and decreased to zero near the wall. However, the velocities near the wall region became slightly large compared with expected velocity value in both cases of water and blood flow X Position [ µ m ] X Position [ µ m ] (a) Water flow (b) Blood flow Fig. 5 Time-averaged velocity distribution in the micro tube in the case of water flow and blood flow at averaged velocity.7 mm/sec In the case of water flow, profiles at both flow rates were symmetry with center of the tube. The results show typical laminar flow as Poiseuille s velocity profile. The theoretical velocity distribution is shown as, r 1 R 2 U ( r) = U c 2 (1) where U (r) is the flow velocity at the position r away from the center of the tube, U c is the velocity at the center and R is the radius of tube. Velocity profiles in the case of blood flow at both flow rates shows typical non-newtonian fluid feature; blunt and broad in the center region and sharp near the wall compared with velocity profiles of water flow. The results were very similar to that of in vivo blood flow in an arteriole (Sugii et al., 22). In blood flow, the viscosity steeply increases by decreasing share rate especially in low shear rate region caused by RBC rheological properties such as deformation and aggregation of RBCs (Maeda et al, 22). However, the obtained velocity seemed that of liquid component of blood, which was a macromolecular protein, not RBC. The results indicate that liquid component of blood also shows flow feature of non-newtonian fluid. 5 Copyright 23 by PSFVIP-4

6 June 3-5, 23, Chamonix, France. F484 Average velocty ( Uave = 3.5 [mm/sec] ) Average velocty ( Uave =.7 [mm/sec] ) Variance ( Uave = 3.5 [mm/sec] ) Variance ( Uave =.7 [mm/sec] ) Theoretical value Normarized velocty u/umax Y Position [pixel] (a) Water flow Average velocity ( Uave = 3.5 mm/sec ) Average velocity ( Uave =.7 mm/sec ) Variance ( Uave = 3.5 mm/sec ) Variance ( Uave =.7 mm/sec ) Normarized velocity u/umax Y Position [pixel] (b) Blood flow Fig. 6 Time-averaged axial velocity profile in the case of water flow and blood flow at averaged velocity.7 mm/sec and 3.5 mm/sec. 6 Copyright 23 by PSFVIP-4

7 June 3-5, 23, Chamonix, France. F Conclusion The micro PIV system was applied to in vitro blood flow through a micro round tube with inner diameter of 1 µm in order to investigate rheological properties of blood flow. The velocity distributions of liquid component such as plasma were measured using blood seeded with.5 µm diameter fluorescent particle. The velocity distributions with spatial resolution of 5.9 x.73 µm were measured even near the wall in the center plane of the round tube. In the case of water seeded with fluorescent particle, the time averaged axial velocity profiles showed nearly parabolic. Contrary, in the case of blood flow, both velocity profiles were blunt at the center region and sharp near the wall. The results indicate that liquid component of blood itself, not RBC, also show flow feature of non- Newtonian fluid. The authors acknowledge Prof. Inada of Gunma University for use of blood. References Bishop J.J., Nance P. R., Popel A.S., Intaglietta M., Johnson P. C., Effect of erythrocyte aggregation on velocity profiles in venules. Am. J. Physiol. Heart. Circ. Physiol., Vol.28, pp , 21. Cochrane T., Earnshaw J. C., Love A.H.G., Laser Doppler measurement of blood velocity in microvessels. Med. Biod. Eng. Comput., Vol.19-15, pp , Gijsen F.J.H., Allanic E., van de Vosse F.N., Janssen J.D., The influence of the non-newtonian properties of blood on the flow in large arteries: unsteady flow in a 9 degree curved tube., Journal of Biomechanics, Vol. 32, pp , Koutsiaris A. K., Mathioulakis D.S., Tsangaris S., Micro scope PIV for velocity-field measurement of particle suspension flowing inside glass capillaries. Meas. Sci. Technol., Vol. 1, pp , Maeda N., Microcirculation of erythrocytes in relation to their rheological properties., Japanese Journal of Nagare, Vol. 21, pp , 22 (in Japanese). Meinhart C. D., Wereley S. T., Santiago J. G.: PIV measurement of microchannel flow, Exp. Fluids, Vol. 27, pp , Okuda R., Sugii Y., Okamoto K., Characterization of high spatial velocity distributions of blood flow obtained by micro PIV technique. CD-ROM Proc. 7 th International Congress Biological and Medical Engineering, Singapore, 22. Sugii Y., Nishio S., Okuno T., Okamoto K., A highly accurate iterative PIV technique using gradient method. Meas. Sci. Technol., Vol.11, pp , 2. Sugii Y., Nakano A., Nishio S., Minamiyama M., Blood flow velocity measurement in microcirculation field by means of highly accurate iterative PIV, Trans. Japan Society of Mechanical Engineers, Vol.67, No.662, pp , 21 (in Japanese). Sugii Y., Nishio S., Okamoto K., In vivo PIV measurement of red blood cell velocity field in microvessels considering mesetery motion, Physiol. Meas., Vol. 23, pp , 22. Wayland H. and Johnson P.C., Erythrocyte velocity measurement in microvessels by two-slit photometric method. J. Appl. Phsiol., Vol. 22, pp , Copyright 23 by PSFVIP-4