Red Blood Cell Velocity and Volumetric Flow Assessment by Enhanced High-Resolution Laser Doppler Imaging in Separate Vessels of the Hamster Cheek Pouch Microcirculation (original) (raw)
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Journal of Biomedical Optics, 2009
The use of laser Doppler perfusion imaging ͑LDPI͒ and laser speckle perfusion imaging ͑LSPI͒ is well known in the noninvasive investigation of microcirculatory blood flow. This work compares the two techniques with the recently developed tissue viability ͑TiVi͒ imaging system, which is proposed as a useful tool to quantify red blood cell concentration in microcirculation. Three systems are evaluated with common skin tests such as the use of vasodilating and vasoconstricting drugs ͑methlynicotinate and clobetasol, respectively͒ and a reactive hyperaemia maneuver ͑using a sphygmomanometer͒. The devices investigated are the laser Doppler line scanner ͑LDLS͒, the laser speckle perfusion imager ͑FLPI͒-both from Moor Instruments ͑Axminster, United Kingdom͒-and the TiVi imaging system ͑Wheels-Bridge AB, Linköping, Sweden͒. Both imaging and point scanning by the devices are used to quantify the provoked reactions. Perfusion images of vasodilatation and vasoconstriction are acquired with both LDLS and FLPI, while TiVi images are acquired with the TiVi imager. Time acquisitions of an averaged region of interest are acquired for temporal studies such as the reactive hyperaemia. In contrast to the change in perfusion over time with pressure, the TiVi imager shows a different response due its measurement of blood concentration rather than perfusion. The responses can be explained by physiological understanding. Although the three devices sample different compartments of tissue, and output essentially different variables, comparisons can be seen between the three systems. The LDLS system proves to be suited to measurement of perfusion in deeper vessels, while FLPI and TiVi showed sensitivity to more superficial nutritional supply. LDLS and FLPI are insensitive to the action of the vasoconstrictor, while TiVi shows the clear boundaries of the reaction. Assessment of the resolution, penetration depth, and acquisition rate of each instrument show complimentary features that should be taken into account when choosing a system for a particular clinical measurement.
An Improved Instrument for Real-Time Measurement of Blood Flow Velocity in Microvessels
IEEE Transactions on Instrumentation and Measurement, 2000
A new approach for the measurement of red blood cell velocity at the level of microcirculation has been developed and characterized. The new real-time and automated measurement system is based on the dual-slit methodology, and blood flow information is extracted from images and transduced into two analog photometric signals and then processed using a hybrid analog-digital system that performs the cross correlation of the signals in real time. The characterization of the system consists of a calibration with a known velocity target, yielding to the hyperbolic calibration curve velocity versus delay and the determination of the velocity detectable range from 0.3 to 120 mm/s. A theoretical study of the measurement uncertainty and parametric studies were carried out to test the system robustness to changes of parameters and to determine the optimal configuration that is applicable to various experimental conditions. The system was further tested in in vivo experiments in the rat cremaster preparation in different types of vessels and flow velocities to verify the consistency of the results, as compared with those from conventional measuring systems. In addition, the dynamic behavior of the system and its response to changes in the measured velocity were studied through a continuous velocity record that was obtained during an experimental procedure.
Red Cell Interactions with the Microcirculation
Microcirculation, 1976
We have measured RBC velocity profiles for mammalian arterioles and venules from high-speed cinematographic motion pictures. Measurements were made at 320x and 400x optical magnification over an averaging time period of 10 ms. In vivo profiles are uniformly nonsymmetrical, the RBCs exhibit rotation, and they frequently deviate sidewise from the overall axial direction of motion. In general, this is more pronounced on the venous side. Since all of the profiles are time variant and the average values are synchronous with the midstream velocity, individual RBC velocities will vary about the average. Profiles become more blunted in vessels with smaller diameters. In vessels below 16 JLm diameter, the velocity gradients between adjacent RBCs are quite small; for large vessels, recognizable profiles develop and become fully developed in blood vessels above 30 JLm in diameter. This blunting is further affected by the midstream velocity and the local hematocrit; when the velocity is reduced below 1.2 mmls and/or an increased hematocrit is present, the profile becomes more blunted. We have estimated the microhematocrit in these blood vessels indirectly by interpolation from systemic and capillary hematocrit distributions and have assumed it to be about 2~30%. Comparable in vitro profile studies indicate the same qualitative effects of hematocrit, velocity, and diameter on the RBC profiles in glass tubes (Bugliarello and Hayden,
Velocity pulse measurements in the mesenteric arterioles of rabbits
Physiological Measurement, 2004
Axial red blood cell velocity pulse was quantified throughout its period by a high speed video microscopy method, using images of erythrocytes moving near the microvessel axis. In 10 mesenteric precapillary arterioles (8 to 12μm in diameter) from 6 rabbits, axial velocities ranged from 0.46 (the minimum of all the end diastolic values) to 4.8mm/s (the maximum of all the peak systolic values). With the velocity pulse shape properly quantified, a correct estimation of the average velocity over time can be made and hence, appropriate quantification of blood flow. Average velocity ranged between 1.14mm/s (8μm arterioles) and 1.98mm/s (9μm arterioles). Also, with the velocity pulse shape known, an estimation of the magnitude of the pulsation can be made by introducing Pourcelot's resistive index (RI) in the microvascular haemodynamics (diameter less than 15μm). The results of this study reveal that RI in the precapillary arterioles is quite high ranging between 0.56 (8μm arterioles) and 0.74 (12μm arterioles). Observing the velocity pulse diagrams in different diameters, quantitative information is obtained for the first time, on how the velocity pulse shape flattens as it proceeds to the capillary bed.
Microvascular Research, 1998
The mean centerline red blood cell (RBC) velocity of the rat pial artery was measured using an image-intensified high-speed (1000 frames/s) video camera system and RBCs labeled with fluorescein isothiocyanate (FITC). Some investigations measuring RBC velocity have been made in most organs, but the RBC velocity of the pial artery has not yet been measured with this system using FITC labeled RBC. After recording the emission of the FITC labeled RBC through a closed cranial window using this system, the authors analyzed the videotape. The movement of each individual RBC for several milliseconds over a distance of 50 m could be pursued. The mean centerline RBC velocity in normal rats varied between 1.0 and 9.0 mm/s (most of the measurements we taken in vessels ranging between 20 and 80 m in diameter). As the diameter of the pial artery becomes smaller, the blood flow rate ( ؋ (diameter/2) 2 ؋ (mean centerline velocity/1.6)) tends to become smaller. During CO 2 inhalation, the pial artery diameter, mean centerline RBC velocity, and blood flow rate increased with statistical significance. Mean centerline RBC velocities in the cerebral microcirculation could not be measured directly with accuracy using the older methods (30 frames/s). However, this method is useful for investigation of the cerebral microcirculation and is considered to be appli-cable for studying the behavior of leukocytes or platelets, which will be examined in a subsequent study.
Velocimetry of red blood cells in microvessels by the dual-slit method: Effect of velocity gradients
Microvascular Research, 2012
The dual-slit is a photometric technique used for the measurement of red blood cell (RBC) velocity in microvessels. Two photometric windows (slits) are positioned along the vessel. Because the light is modulated by the RBCs flowing through the microvessel, a time dependent signal is captured for each window. A time delay between the two signals is obtained by temporal cross correlation, and is used to deduce a velocity, knowing the distance between the two slits. Despite its wide use in the field of microvascular research, the velocity actually measured by this technique has not yet been unambiguously related to a relevant velocity scale of the flow (e.g. mean or maximal velocity) or to the blood flow rate. This is due to a lack of fundamental understanding of the measurement and also because such a relationship is crucially dependent on the non-uniform velocity distribution of RBCs in the direction parallel to the light beam, which is generally unknown. The aim of the present work is to clarify the physical significance of the velocity measured by the dual-slit technique. For that purpose, dual-slit measurements were performed on computer-generated image sequences of RBCs flowing in microvessels, which allowed all the parameters related to this technique to be precisely controlled. A parametric study determined the range of optimal parameters for the implementation of the dual-slit technique. In this range, it was shown that, whatever the parameters governing the flow, the measured velocity was the maximal RBC velocity found in the direction parallel to the light beam. This finding was then verified by working with image sequences of flowing RBCs acquired in PDMS micro-systems in vitro. Besides confirming the results and physical understanding gained from the study with computer generated images, this in vitro study showed that the profile of RBC maximal velocity across the channel was blunter than a parabolic profile, and exhibited a non-zero sliding velocity at the channel walls. Overall, the present work demonstrates the robustness and high accuracy of the optimized dual-slit technique in various flow conditions, especially at high hematocrit, and discusses its potential for applications in vivo.
2010
Not all tumor vessels are equal. Tumor-associated vasculature includes immature vessels, regressing vessels, transport vessels undergoing arteriogenesis and peritumor vessels influenced by tumor growth factors. Current techniques for analyzing tumor blood flow do not discriminate between vessel subtypes and only measure average changes from a population of dissimilar vessels. We have developed methodologies for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at single capillary resolution in vivo. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope and provides fully-resolved three-dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are asymmetric near intussusceptive tissue structures in tumors in mice. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in, around a tumor and in normal brain. + Correspondence: munn@steele.mgh.harvard.edu (L.L.M.). Author Contributions W.S.K., Conception and design, technique validation, implementation and data collection, manuscript writing and final approval of manuscript; S.S.C, implementation and data collection; D.A.L, implementation and data collection; J.A.T., conception and design and technique validation; M.M., technique validation; M. A. G., implementation and data collection; D.F., conception and design and administrative support; R.K.J, administrative support, financial support and final approval of manuscript; L.L.M, Conception and design, administrative support, financial support, manuscript writing and final approval of manuscript.
Methods to measure blood flow velocity of red blood cells in vivo at the microscopic level
Annals of Biomedical Engineering, 1986
Several methods to measure red blood cell velocity in microvessels by electronic means are discussed. Signals are generated by the red blood cells present in the microscopic image of the microvessels. These signals can be converted to obtain an output signal proportional tO the actual red blood cell velocity. The method of spatial filtering by interlacing gratings is discussed in terms of a filter with an input signal. Adaptation of optical factors that might improve the velocity measurement is obtained by a mathematical analysis. Different methods of correlation are presented. The temporal correlation (dual slit and video window) and spatial correlation methods are discussed in relation to factors influencing the quality of the correlogram, the peak of which is proportional to red blood cell velocity. The conversion o fred blood cell velocity to volume flow is put in perspective.
Determination of Red Blood Cell Velocity by Video Shuttering and Image Analysis
Annals of Biomedical Engineering, 2000
A novel modification of conventional video imaging techniques has been developed to determine the velocity of red blood cells ͑RBCs͒, which offers compatibility with existing video-based methods for determining blood oxygenation and hemoglobin concentration. Traditional frame-by-frame analysis of video recordings limits the maximum velocity that can be measured for individual cells in vivo to about 2 mm/s. We have extended this range to about 20 mm/s, by electronic shuttering of an intensified charge-coupled device camera to produce multiple images of a single RBC in the same video frame. RBCs were labeled with fluorescein isothiocyanate and the labeled cells ͑FRBCs͒ were used as probes to determine RBC velocities in microvessels of the hamster retractor muscle. Velocity was computed as the product of the distance between centroids of two consecutive image positions of a FRBC and the shuttering frequency of the camera intensifier. In vitro calibrations of the system using FRBC and Sephadex beads coated onto a rotating disk yielded an average coefficient of variation of about 6%. Flow conservation studies at bifurcations indicated that the maximum diameter of microvessels below which all the FRBCs in the lumen could be detected was 50 m. The technique was used to estimate mean-FRBC velocity distributions in vessels with diameters ranging from 8 to 50 m. The mean-FRBC velocity profiles were found to be blunter than would be expected for Poiseuille flow. Single FRBCs tracked along an unbranched arteriole exhibited significant temporal variations in velocity. © 1999 Biomedical Engineering Society. ͓S0090-6964͑99͒02103-7͔