In addition to structural imaging, OCT can be used for Doppler analysis to obtain velocity measurements from moving structures, with the same spatial and temporal resolution [39
]. Doppler OCT relies on detection of a phase shift between adjacent in-depth OCT scans at each point, which is caused by movement of the light scatterers. Blood flow velocity at each pixel can be reconstructed according to the formula (11):
where Δϕ - is a Doppler shift-induced phase shift calculated between successive A-scans, n - is a refractive index, <k> - is the average wave number, τ - is time between the successive A-scans, and β - is an angle between the flow direction and the laser beam. The angle β can be calculated from structural 2-D and 3-D data sets.
Doppler OCT is an effective way to characterize blood flow dynamics in early embryos analysis. It can be applied to reconstruct spatially and temporally resolved Doppler shift velocity profiles from yolk sac vessels and embryonic vessels when the blood flow is well established [35
], as well as at early stages of circulation while blood flow is being established, based on velocity measurements from individual circulating blood cells [34
]. This is highly important as it allows visualizing earliest blood circulation defects in mouse mutants with cardiovascular abnormalities, which is currently not possible with other modalities.
Doppler OCT imaging in rat embryos has also been performed [40
]. shows an example of a structural OCT image through the E10.5 rat embryonic heart and a series of color-coded Doppler images of the same area taken from a time lapse and representing different phases of the heartbeat cycle. The Doppler shift signal is generated by the velocity component, which is parallel to the OCT laser beam. The blue shift corresponds to movement toward the detector, while the red shift corresponds to movement away from the detector. As one can see from the figure, the Doppler shift was detected from the circulating blood cells inside the heart (labeled as b) as well as from the moving heart wall (labeled as hw).
Figure 2 Doppler OCT velocity imaging in the live rat embryonic heart at 10.5 dpc. (A) Structural image acquired from the primitive ventricle of the beating embryonic heart. (B–G) Corresponding representative Doppler color-coded maps from the same area (more ...)
In many cases, hemodynamic analysis in the heart is complicated by the Doppler shift phase wrapping (at high flow velocities, the Doppler OCT shift exceeds 2π, which makes velocity calculations ambiguous) and the difficulty of mapping the exact flow direction, which is required for the velocity calculation. Several groups are developing and optimizing algorithms to overcome these limitations in avian models [26
]. Potentially, these methods can be applied for quantitative Doppler OCT hemodynamic analysis in mammalian embryonic hearts.
Traditionally, the vascular structure is reconstructed based on Doppler OCT analysis of the blood flow. The major drawback of Doppler OCT for blood flow analysis and reconstruction of vascular structure is its insensitivity to the transverse component of blood flow, which prevents Doppler OCT visualization of vessels perpendicular to the scanning laser beam. Another disadvantage of the Doppler OCT is its dependence on phase stability of the system. Alternatively, Speckle Variance (SV) OCT analysis can be used for 3-D reconstruction of the vasculature in cultured embryos [41
]. SV OCT analysis relies on statistical properties of time-varying speckle pattern in OCT images, as the decorrelation of speckles from moving scatterers is faster than that of static scatterers. Because the most dynamic scatterers in the embryo are circulating blood cells, this algorithm allows to visualize 3-D circulatory network. SV OCT imaging provides more accurate 3-D visualization of the vasculature than the Doppler OCT method, since Doppler OCT relies on the axial component of the blood flow and SV OCT it is not sensitive to the flow direction.