Chaminda R. Samarage

In Vivo Wall Shear Measurements within the Developing Zebrafish Heart

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.

Quantification of heterogeneity in lung disease with image-based pulmonary function testing

Computed tomography (CT) and spirometry are the mainstays of clinical pulmonary assessment. Spirometry is effort dependent and only provides a single global measure that is insensitive for regional disease, and as such, poor for capturing the early onset of lung disease, especially patchy disease such as cystic fibrosis lung disease. CT sensitively measures change in structure associated with advanced lung disease. However, obstructions in the peripheral airways and early onset of lung stiffening are often difficult to detect. Furthermore, CT imaging poses a radiation risk, particularly for young children, and dose reduction tends to result in reduced resolution. Here, we apply a series of lung tissue motion analyses, to achieve regional pulmonary function assessment in β-ENaC-overexpressing mice, a well-established model of lung disease. The expiratory time constants of regional airflows in the segmented airway tree were quantified as a measure of regional lung function. Our results showed marked heterogeneous lung function in β-ENaC-Tg mice compared to wild-type littermate controls; identified locations of airway obstruction, and quantified regions of bimodal airway resistance demonstrating lung compensation. These results demonstrate the applicability of regional lung function derived from lung motion as an effective alternative respiratory diagnostic tool.

Assessment of airway response distribution and paradoxical airway dilation in mice during methacholine challenge

Detailed information on the distribution of airway diameters during bronchoconstriction in situ is required to understand the regional response of the lungs. Imaging studies using computed tomography (CT) have previously measured airway diameters and changes in response to bronchoconstricting agents, but the manual measurements used have severely limited the number of airways measured per subject. Hence, the detailed distribution and heterogeneity of airway responses are unknown. We have developed and applied dynamic imaging and advanced image-processing methods to quantify and compare hundreds of airways in vivo. The method, based on CT, was applied to house dust-mite-sensitized and control mice during intravenous methacholine (MCh) infusion. Airway diameters were measured pre- and post-MCh challenge, and the results compared demonstrate the distribution of airway response throughout the lungs during mechanical ventilation. Forced oscillation testing was used to measure the global response in lung mechanics. We found marked heterogeneity in the response, with paradoxical dilation of airways present at all airway sizes. The probability of paradoxical dilation decreased with decreasing baseline airway diameter and was not affected by pre-existing inflammation. The results confirm the importance of considering the lung as an entire interconnected system rather than a collection of independent units. It is hoped that the response distribution measurements can help to elucidate the mechanisms that lead to heterogeneous airway response in vivo.NEW & NOTEWORTHY Information on the distribution of airway diameters during bronchoconstriction in situ is critical for understanding the regional response of the lungs. We have developed an imaging method to quantify and compare the size of hundreds of airways in vivo during bronchoconstriction in mice. The results demonstrate large heterogeneity with both constriction and paradoxical dilation of airways, confirming the importance of considering the lung as an interconnected system rather than a collection of independent units.

Optimisation of a Stirred Bioreactor through the Use of a Novel Holographic Correlation Velocimetry Flow Measurement Technique

We describe a method for measuring three dimensional (3D) velocity fields of a fluid at high speed, by combining a correlation-based approach with in-line holography. While this method utilizes tracer particles contained within the flow, our method does not require the holographic reconstruction of 3D images. The direct flow reconstruction approach developed here allows for measurements at seeding densities in excess of the allowable levels for techniques based on image or particle reconstruction, thus making it suited for biological flow measurement, such as the flow in bioreactor. We outline the theory behind our method, which we term Holographic Correlation Velocimetry (HCV), and subsequently apply it to both synthetic and laboratory data. Moreover, because the system is based on in-line holography, it is very efficient with regard to the use of light, as it does not rely on side scattering. This efficiency could be utilized to create a very high quality system at a modest cost. Alternatively, this efficiency makes the system appropriate for high-speed flows and low exposure times, which is essential for imaging dynamic systems.

Technical Note: Contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source

Purpose: In vivo imaging of the pulmonary vasculature in small animals is difficult yet highly desirable in order to allow study of the effects of a host of dynamic biological processes such as hypoxic pulmonary vasoconstriction. Here the authors present an approach for the quantification of changes in the vasculature. Methods: A contrast free angiography technique is validated in silico through the use of computer-generated images and in vivo through microcomputed tomography (μCT) of live mice conducted using a laboratory-based x-ray source. Subsequent image processing on μCT data allowed for the quantification of the caliber of pulmonary vasculature without the need for external contrast agents. These measures were validated by comparing with quantitative contrast microangiography in the same mice. Results: Quantification of arterial diameters from the method proposed in this study is validated against laboratory-based x-ray contrast microangiography. The authors find that there is a high degree of correlation (R = 0.91) between measures from microangiography and their contrast free method. Conclusions: A technique for quantification of murine pulmonary vasculature without the need for contrast is presented. As such, this technique could be applied for longitudinal studies of animals to study changes to vasculature without the risk of premature death in sensitive mouse models of disease. This approach may also be of value in the clinical setting.

Novel Analysis of 4DCT Imaging Quantifies Progressive Increases in Anatomic Dead Space During Mechanical Ventilation in Mice

Increased dead space is an important prognostic marker in early acute respiratory distress syndrome (ARDS) that correlates with mortality. The cause of increased dead space in ARDS has largely been attributed to increased alveolar dead space due to ventilation/perfusion mismatching and shunt. We sought to determine whether anatomic dead space also increases in response to mechanical ventilation. Mice received intratracheal lipopolysaccharide (LPS) or saline and mechanical ventilation (MV). Four-dimensional computed tomography (4DCT) scans were performed at onset of MV and after 5 h of MV. Detailed measurements of airway volumes and lung tidal volumes were performed using image analysis software. The forced oscillation technique was used to obtain measures of airway resistance, tissue damping, and tissue elastance. The ratio of airway volumes to total tidal volume increased significantly in response to 5 h of mechanical ventilation, regardless of LPS exposure, and airways demonstrated significant variation in volumes over the respiratory cycle. These findings were associated with an increase in tissue elastance (decreased lung compliance) but without changes in tidal volumes. Airway volumes increased over time with exposure to mechanical ventilation without a concomitant increase in tidal volumes. These findings suggest that anatomic dead space fraction increases progressively with exposure to positive pressure ventilation and may represent a pathological process.NEW & NOTEWORTHY We demonstrate that anatomic dead space ventilation increases significantly over time in mice in response to mechanical ventilation. The novel functional lung-imaging techniques applied here yield sensitive measures of airway volumes that may have wide applications.

Polynomial element velocimetry (PEV): a technique for continuous in-plane velocity and velocity gradient measurements for low Reynolds number flows

Particle image velocimetry (PIV) selects the maximum of the cross-correlation map to represent the modal displacement, and a wealth of information stored in the cross-correlation is discarded. We introduce a novel method, termed polynomial element velocimetry (PEV), which results in continuous velocity and velocity gradient measurements. PEV utilizes the extra information stored in the cross-correlation to determine continuous velocity measurements with low levels of measurement noise. In contrast to PIV, the continuous nature of velocity measurements facilitates the direct determination of the velocity gradient. The PEV method is applied to two laboratory flows: flow in a channel and flow behind a circular cylinder at Reynolds number, Re?= 30, and is shown to greatly reduce the noise in the measurements. In addition, the accuracy of PEV is validated using two computer-generated synthetic flows: parabolic flow in a channel and flow past a square cylinder at Re?= 30. In these cases, PEV is shown to reduce the velocity measurement error by up to 45% and the vorticity estimation error by up to 77% when compared to PIV. A key benefit of the PEV method is that it is capable of calculating continuous measures for flow gradient with greatly reduced bias errors. In particular, PEV provides a more accurate measurement of the vorticity near interfaces such as a cylinder wall or channel wall where PIV methods only provide measurement data at half the sampling window size from the wall. Since PEV utilizes the entire shape of the cross-correlation map to determine a local map for the underlying velocity, minimal random error is transmitted to the estimated flow gradient. This feature of the PEV method makes it optimal for flows where flow gradients are well defined and there are insufficient pixels to fully resolve structures in the flow using PIV.

Application of a novel diffraction-based tomography method for imaging biological samples

A novel system to image and reconstruct a 3-dimensional map of the refractive index based on the diffraction of light through a transparent sample is presented. This method is tested and validated on computer-generated data sets. The proposed system is an advanced variation of an imaging technique used in engineering for the study of aerodynamics. This method, which is termed Reference Image Topography, is used to reconstruct the water/air interface of the free surface in fluid dynamics studies. This surface profile is reconstructed by comparing an image of a random pattern viewed through the transparent free surface against a reference image, to determine the change in the refractive index caused by changes in the height. The proposed system is highly sensitive and capable of imaging intricate features in the transparent sample that are of low contrast when imaged with other imaging methods. For each projection, the change in direction of the light passing through the sample when placed in between the light source and the imaging system, can be related to the line integral for the change in refractive index across the sample. Utilizing multiple projections, a 3- dimensional map of the refractive index of the sample is reconstructed with computed tomography.