An x-ray velocimetry technique is described which provides three components of velocity measurement in three dimensional space. Current x-ray velocimetry techniques, which use particle images taken at a single projection angle, are limited to two components of velocity measurement, and are unable to measure in three dimensions without a priori knowledge of the flow field. The proposed method uses multiple projection angles to overcome these limitations. The technique uses a least-squares iterative scheme to tomographically reconstruct the three-dimensional velocity field directly from two-dimensional image pair cross-correlations, without the need to reconstruct three-dimensional particle images. Synchrotron experiments demonstrate the effectiveness of the technique for blood flow measurement in opaque vessels, with applications for the diagnosis and treatment of cardiovascular disease.

Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.

High resolution in vivo velocity measurements within the cardiovascular system are essential for accurate calculation of vessel wall shear stress, a highly influential factor for the progression of arterial disease. Unfortunately, currently available techniques for in vivo imaging are unable to provide the temporal resolution required for velocity measurement at physiological flow rates. Advances in technology and improvements in imaging systems are allowing a relatively new technique, X-ray velocimetry, to become a viable tool for such measurements. This study investigates the haemodynamics of pulsatile blood flow in an optically opaque in vitro model at physiological flow rates using X-ray velocimetry. The in vitro model, an asymmetric stenosis, is designed as a 3:1 femoral artery with the diameter and flow rate replicating vasculature of a mouse. Velocity measurements are obtained over multiple cycles of the periodic flow at high temporal and spatial resolution (1 ms and 29 μm, respectively) allowing accurate measurement of the velocity gradients and calculation of the wall shear stress. This study clearly illustrates the capability of in vitro X-ray velocimetry, suggesting it as a possible measurement technique for future in vivo vascular wall shear stress measurement.

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at delivery into mouse trachea via immediate retrograde fluid motion, and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways.These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small animal models.

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