In vivo Doppler and three-dimensional OCT images were acquired from three blood vessels in an HH 17 chick embryo. contains a normalized color Doppler OCT image of blood flow through the cross-section of Vessel 2 superimposed over a B-mode OCT structural image. The red or blue of the DOCT image corresponds to blood flow in the direction towards or away from the incident OCT beam. The intensity of the grayscale structural image correlates to the reflectivity of the microstructures in the tissue. The dotted line in indicates the A-scan location of SDV measurement. SDV measurements were taken along the center of all three vessels as illustrated in . is a volumetric rendering of Vessel 2 (purple) with two orthogonal OCT cross-sectional images. The x-y plane pinpoints the location of the SDV measurement within the 3D vessel structure. This plane is the same as . The angle of flow relative to the OCT beam is measured using similar volume renderings for all three vessels.
Blood flow velocity dynamics and the spatial velocity profile of the three vessels are shown in . An example Doppler M-mode (depth – y-axis vs. time – x-axis) image from Vessel 2 is shown in . As in , the normalized color Doppler is superimposed over OCT A-scans collected over time, from the same location in the vessel. A plot of the Doppler measurement from the center of the vessel as a function of time provides information on the blood flow dynamics in the vessel (). Velocity as a function of time in each vessel was calculated using Equation (2)
. This plot provides time-resolved velocity measurements of blood flow through Vessel 1 (red), Vessel 2 (green), and Vessel 3 (blue). The initial time for each measurement was arbitrarily chosen at a point when the velocity was near zero. These plots show the increase in velocity as blood passes through the SDV line of interrogation. Peak velocities were approximately 3.1, 2.0, and 8.0 mm/s for Vessel 1, Vessel 2, and Vessel 3, respectively; and the velocity drops to zero at times correlating to diastole. The blood velocity rates are on the order of those measured using micro particle image velocimetry (Lee et al., 2007
). These results are also consistent with the expectation that peak blood flow velocities decrease in peripheral vessels further downstream from the heart. The vitelline vessel (Vessel 3) is a major blood vessel that connects to the dorsal aorta and where we measured velocity flow rates over 2.5 times faster than the other two, more peripheral vessels. In each case there is also a small decrease in velocity that occurs during peak flow. This transient decrease in flow may represent the dicrotic notch and wave that is observed in post-embryonic peripheral arteries (Troxler and Wilkinson, 2007
) (see discussion).
Figure 5 Blood flow measurements from three extraembryonic vessels. (a) Depth (y-axis) vs. time (x-axis) Doppler M-mode (blue) superimposed over M-mode OCT scans of Vessel 2. (b) SDV measurement taken along the dotted horizontal line in (a) shows the blood flow (more ...)
As previously mentioned, an inherent advantage of SDV over pulsed wave Doppler ultrasound is the ability to acquire depth resolved velocity measurements. A plot of the blood flow velocity through the diameter of each vessel is shown in . These velocity profiles were sampled at a time near peak flow through the vessels (t = ~110 ms for Vessel 1 and 2, t = ~145 ms for Vessel 3).
One challenge in resolving the blood flow profiles is that the high optical attenuation of blood reduces optical contrast in OCT images. This is best demonstrated in , where a “shadow” appears below the blood vessel. This shadow can also add additional phase noise to Doppler images in the same region. As a result, accurately measuring blood flow in vessels that are large or reside deeper in tissue may prove difficult. The asymmetry of the blood flow profile from Vessel 3 was most likely caused by optical attenuation near the bottom of the vessel. The shear rate on the vessel wall was based solely on the ascending slope of the velocity profile. The calculated shear rates for Vessels 1, 2, and 3 were 54.2, 74.5, and 25 s−1, respectively. The table in outlines the measured diameter and volumetric flow rate from each vessel.
This investigation of extraembryonic vessels was necessary to develop the technology for interpreting flow through the more complex and dynamically beating heart tube. demonstrates preliminary proof-of concept measurement of blood flow through the outflow tract of an HH 16 chicken embryo heart. SDV measurements were acquired along the center of the outflow tract, as indicated by the dotted line in . The active pumping of the heart tube produces a more complex temporal blood flow profile than observed in the extraembryonic vessels. shows the blood flow velocity through the outflow tract, as a function of time, during one heart beat cycle. Using the M-mode OCT image in , the blood flow velocity can be correlated to the diameter of the outflow tract during ejection of blood, this data could be used to measure the volumetric flow rate. Peak blood flow during the cycle reached approximately 18 mm/s, which is within the range of measurements reported using pulsed Doppler ultrasound ((McQuinn et al., 2007
): outflow velocity ~14.3 mm/s for HH 24 chick), pulsed Doppler velocimetry ((Hu and Clark, 1989
): peak dorsal aorta velocity increases from ~30 mm/s to ~40 mm/s from HH 12 to HH 24 in development), and micro particle velocimetry ((Vennemann et al., 2006
):peak primitive ventricle velocity ~25 mm/s in HH 15 chick embryo). To our knowledge, there is no reported data on blood flow velocities for HH 17 chick embryos measured in the outflow tract region of the heart tube. These preliminary results suggest that there is negative, or regurgitant, flow that occurs while the outflow tract is open, possibly due to incomplete formation of the endocardial cushions. The spatial blood flow velocity profile during peak flow (vertical dotted line in ) is shown in . These results are preliminary in nature but they successfully demonstrate the ability to use OCT and SDV to image and measure depth-resolved blood flow, non-invasively in these very early stage chicken embryo heart tubes.