Fig. 1. Gated Reconstruction. The figure depicts B-scans captured for 4 different phases in the cardiac cycle before moving to the next position to capture B-scans in the same phase as in the previous position. 3D images are reconstructed at each phase.

Fig. 2. Gated OCT Setup. Arbitrary waveform generators control timing elements (scanning optics and frame synchronization signal) in the OCT setup. We paced the heart at 1 Hz and are capable of capturing 4800 B-scans in five minutes. AWG 1 - arbitrary waveform generator 1, AWG 2 - arbitrary waveform generator 2.

Fig. 3. Three-dimensional reconstruction of a 13.5 dpc embryonic mouse heart, ventral view (top left panel). A digital image of the same mouse heart is below the rendered image. The scale bar applies to all of the images. Panels A-D are samples of transverse 2D OCT “slices” through the heart at levels represented by the labeled lines in the reconstruction. The smooth walled outflow tract (oft) can be distinguished from the trabeculated right and left ventricles (panels B and C) right ventricle - RV, left ventricle - LV. The “corrugated” inner surfaces of the atria are also visible (panels A and B, left and right atria: LA and RA)

Fig. 4. (A) Digitally “sliced” open embryonic mouse heart (same heart as in Figure 3). The scale bar applies to all of the images. The grey regions represent the “cut” surface, the red areas are the reconstructed portion. The rough ventricular trabecular surface can be easily distinguished from the smooth endocardial surface of the atria, and endocardial cushions lining the lumen are easily seen. Note that the OCT slices used to create the reconstruction in Figure 3 were transversely oriented, however once the reconstruction is made, the heart can be viewed from any angle, section, or view. (B) (321 KB) Three-dimensional reconstruction of a 13.5 dpc embryonic mouse heart, ventral view. The black line indicates the slice for the heart in (A) [Media 1].

Fig. 5. Stage 13 chick embryo heart. All images in panel come from the same heart except for panel H and I. (A) Photograph of heart taken after fixation. (B) (752 KB) 3D OCT image taken with the 4D-OCT setup, providing a ‘snapshot’ of one of the phases in the cardiac cycle [Media 2]. (C & D) (370 KB) Embryonic chick heart in end-relaxation and end-contraction obtained with the 4D-OCT scanner. The yellow arrows point to a closed then open OFT [Media 3]. (E & F) Scanning micrographs cut with the same orientation as the 2D scans in panel G. The arrows point to the endothelial invaginations. (G) (567 KB) Two 2D images from the same position (position 16), but different phases (end-relaxation and end-contraction) in the cardiac cycle [Media 4]. Outlines delineate the ventricular chamber (smaller at end-contraction). The yellow outline indicates area used in the calculation of ejection fraction. The yellow arrow points to trabeculae in the ventricle. (H & I) Sections through the embryonic heart stained with H&E (panel H & I show low and high power images of the same section, respectively) and anti-sarcomeric actin (panel I, red). The unlabeled arrow points to the endothelial invagination, absent in panel I. CM -compact myocardium, CJ - cardiac jelly, D - dorsal, V - ventral, Pos 16 - position 16, Vent - ventricle, OFT - outflow tract, End-Relax - end-relaxation, and End-Cont - end-contraction.

Fig. 6. Top panel: 3D reconstruction of a stage 15 embryonic chick heart and a corresponding subset of 2D OCT images used in its construction (A-D). Bottom panel: 3D reconstruction of a stage 20 embryonic chick heart and a corresponding subset of 2D OCT images used in its construction (E-H). Panels A-D: The red outline marks endocardium of the heart tube that is in the process of contraction, green lines indicate that part of the heart tube in the process of relaxation. Panels E-H: The outflow tract cushions (asterisks in panel E) can easily be distinguished, as can the atrioventricular endocardial cushions (EC in panel G). The interventricular foramen between the primitive left and right ventricles can also be distinguished from the atrioventricular cushions (panel F). The scale bar applies to all of the images. oft - outflow tract, IVF - interventricular foramen, EC - endocardial cushions, LA - left side of atrium, LV - left ventricle, RV - right ventricle.

Fig. 7. Wall thickness measured in a stage 15 embryonic heart. Panel A indicates the technique employed (see text). Panel B graphically shows the wall thickness during relaxation of contraction for the same heart at the radial positions indicated in Panel A.

Fig. 8. (337 KB) Peristalsis in a stage 13 embryonic chick heart is demonstrated in these selected OCT slices. The heart (same one as in Fig. 5) was originally imaged in the transverse direction. After the reconstruction was made, sections through the ventricle (vent) and outflow tract (oft) were digitally prepared. This orientation permitted the very first visualization of peristalsis as seen from the endocardial surface. The red outline traces the endocardial surface. The heart is paced just prior to image 1, and the fluid within the heart is distributed evenly between the ventricle and the outflow tract (arrows) until image 3, when the bolus is ejected into the outflow tract (3 and 4). Image 4 has been termed “end-contraction”. Image 10 reflects “end-relaxation”, and the fluid bolus is primarily within the ventricle. [Media 5]