IEEE TRANSACTIONS ON INSTRUMENTATION AND MEASUREMENT, VOL. 56, NO. 6, DECEMBER 2007 2663 An Improved Instrument for Real-Time Measurement of Blood Flow Velocity in Microvessels Francesca Sapuppo, Student Member, IEEE, Maide Bucolo, Member, IEEE, Marcos Intaglietta, Paul C. Johnson, Luigi Fortuna, Fellow, IEEE, and Paolo Arena, Senior Member, IEEE Abstract—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 deter- mination of the velocity detectable range from 0.3 to 120 mm/s. A theoretical study of the measurement uncertainty and para- metric 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 conven- tional 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. Index Terms—Cross correlation, dual-slit methodology, dy- namic measurements, in vivo experimentation, microcirculation, red blood cells (RBCs). I. I NTRODUCTION T HE DEVELOPMENT of automatic real-time methods to determine microvascular functional parameters, such as red blood cell (RBC) flow velocity, has been an active area of development in the microcirculation research field. These methods find their application in the diagnostics of pathologies such as retinal abnormalities, hypertension, and Manuscript received May 30, 2005; revised May 17, 2007. This work was supported in part by the Italian “Ministero dell’Istruzione, dell’Università e della Ricerca” (MIUR) funded project for the Internationalization of the Ph.D. School in Electronics and Automation of the University of Catania, Catania, Italy, and in part by the U.S. Public Health Service (USPHS) under Bioengineering Research Partnership Grant R24-HL64395. The work of M. Intaglietta was supported by the USPHS under Grant R01-HL62354 and Grant R01-HL62318. The work of P. C. Johnson was supported by the USPHS under Grant R01-HL66318. F. Sapuppo, M. Bucolo, L. Fortuna, and P. Arena are with the Dipartimento di Ingegneria Elettrica Elettronica e dei Sistemi, University of Catania, 95125 Catania, Italy (e-mail: fsapuppo@diees.unict.it; mbucolo@diees.unict.it; lfortuna@diees.unict.it; parena@diees.unict.it). M. Intaglietta and P. C. Johnson are with the Department of Bioengineer- ing, University of California, San Diego, La Jolla, CA 92093 USA (e-mail: mintagli@ucsd.edu; pjohnson@bioeng.ucsd.edu). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TIM.2007.907959 cancer, which can be characterized through the analysis of microvascular conditions that involve either angiogenic phe- nomena or functional changes in microcirculation. Moreover, current experimental studies that are associated with the devel- opment of artificial blood [1], [2] require information on blood flow behavior and the development of an analytical framework with which the consequences of altering physical properties of the blood can be analyzed [3]. The first step for the modeling of such a complex envi- ronment consists of objective measurements of structural and functional parameters in microcirculation. Suitable experimen- tal models such as the hamster window model or rat preparation provide a direct visual/optical access to the microcirculation in vivo, offering parts of the body where the skin is thin and extensible. Automated measurement methods are desirable for obtaining this type of information since manual operator-based measure- ments are prone to operator errors, are time consuming, and are potentially nonobjective. Various blood velocity measurement experimental tech- niques exist for the microcirculation environment. The ultrasonic Doppler flowmeter can be applied to small blood vessels, but contrast agents have to be used to meet the spatial resolution requirements for microcirculation appli- cations. Several studies about interaction of ultrasound contrast agents with microcirculation assess that they might interfere with the normal activity due to their interaction with microves- sel lumen [4] and extra-cellular structures [5], thus causing disruption, displacement, separation of endothelial cells, or a phenomenon known as inertial cavitation, leading to bubble destruction [6]–[8]. An alternative technique that is suitable for microcirculation studies is the optical Doppler intravital velocimeter. It can be used in microcirculatory vessels of all sizes, but there are prob- lems in measuring flow in capillaries with tissues having two or more layers of capillaries since the signal is obtained from deeper vessels, as well as the superficial one that is in view. However, this is not a problem with arterioles and venules. Other limitations are that low light levels, tissue movements, and electronic noise can lead to erroneous readings [9]. It is also important to consider that methods based on frequency analyses present sensitivity to any periodic physiological phenomena interfering with the experimental setup, such as blood flow fluctuation due to the heartbeat or respiration [10]. An improved technique based on the Doppler principle is the enhanced high-resolution laser Doppler imaging (EHR- LDI). It is based on the principle of light scattering and on the 0018-9456/$25.00 © 2007 IEEE